Executive Summary 

Maintaining testosterone within a defined physiological range during testosterone replacement therapy (TRT) is often viewed as a straightforward goal. While intuitive, this approach can be difficult to achieve across testing methods, particularly with widely prescribed transdermal forms (gels and creams), where serum and saliva testing results diverge. Although serum testing is the evidence-based standard among traditional providers, perspectives on testing methods for TRT monitoring remain mixed among integrative/functional medicine providers. Saliva testing in particular has gained popularity without demonstrated clinical validity for transdermal TRT monitoring. To address this issue, a recently published review of research studies, clinical guidelines, and real-world lab data evaluates how well different testing methods reflect transdermal TRT dosing and clinical outcomes. While this is a review article, it also included original data showing that hundreds of women taking transdermal testosterone and presenting with elevated salivary testosterone levels did not report more high-androgen symptoms than women without high salivary testosterone levels. The publication’s key findings include:

• Serum testosterone correlates with clinical outcomes. Changes in serum testosterone during transdermal TRT consistently align with changes in bone mineral density, lean body mass, sexual function, hematologic parameters (erythrocytosis), endocrine feedback (luteinizing hormone suppression), and androgen-excess symptoms.
• Clinical effects require raising physiological serum levels. Clinical effects are observed only when serum testosterone levels are raised into the normal range (typically using standard transdermal TRT doses of 25-100mg gel in men or 5-10mg gel or cream in women). When serum testosterone levels do not adequately increase, clinical effects are not observed.  
• Saliva testing shows exaggerated responses. The same transdermal TRT doses that result in normal serum levels routinely produce supraphysiological saliva testosterone values, often interpreted by integrative providers as evidence of excessive dosing. This interpretation leads to selection of doses lower than those demonstrated to have clinical efficacy. 
• Elevated saliva levels lack clinical correlation. No published studies show that the elevated saliva responses to transdermal TRT are accompanied by corresponding changes in any clinical outcomes or symptoms. New data confirms this in hundreds of women on transdermal TRT. Collectively, the evidence suggests that saliva does not reflect tissue-level hormone activity during transdermal TRT.  
• Dried bloodspot (DBS) testing shows similar responses as saliva, with supraphysiological responses to transdermal TRT that lack clinical correlation, limiting its utility for monitoring. 
• Urine testing may complement serum. Urine testosterone levels generally correlate with serum levels during transdermal TRT but should be used as an adjunct to obtain complementary information on androgen metabolism, rather than as a primary monitoring tool.

Conclusions: Serum testing remains the most validated method for monitoring transdermal TRT. Saliva testing is not supported as a serum surrogate, as its results do not reflect TRT dosing and clinical outcomes. Making TRT dosing decisions based on supraphysiological saliva values may lead to underdosing and missed therapeutic benefits.

Keeping testosterone levels within the normal range is paramount for ensuring safety and achieving the clinical benefits of testosterone replacement therapy (TRT). Ideally, this is achieved simply by measuring testosterone levels and adjusting the TRT dose to keep levels within the normal range, no matter which testing method is used. In practice, however, careful selection of a testing method appropriate for the therapy route of administration is critical. This is because different testing methods (namely, saliva and serum) can yield wildly different testosterone results, particularly when using the widely prescribed transdermal route of administration (testosterone gels and creams). These differences matter because they ultimately affect dosing decisions.

While blood (serum) testing is considered the gold standard for measuring testosterone levels during TRT monitoring, saliva testing has emerged as a popular alternative among integrative/functional medicine providers, largely due to its convenient, noninvasive collection. However, the rise in saliva testing use happened without establishing its clinical validity for TRT monitoring. Saliva testing has been shown to be reliable and effective for cortisol measurement in particular, but its validity has not been demonstrated for sex hormone measurement, including testosterone, in patients on transdermal TRT. 

A new peer-reviewed article published in Frontiers in Reproductive Health takes a deep dive into this issue. Authored by the Precision Analytical research team, the article’s clinically relevant conclusions about which testing methods best reflect clinical reality are based on an in-depth review of peer-reviewed research (over 100 published studies), clinical guidelines, and real-world evidence from laboratory databases. The focus is on saliva and serum testing but also touches on dried bloodspot (DBS) and urine testing during transdermal TRT. At its core, this publication is about clinical decision making. The goal of this work was not to take a position for or against any testing method, but to examine how well different testing approaches support effective, evidence-based care for patients on transdermal TRT.  

Serum testosterone aligns with clinical outcomes during transdermal TRT

Our review of the evidence reinforced that serum testosterone measurement remains the most validated and clinically supported method for monitoring transdermal TRT. Numerous studies have reported that serum testosterone concentrations rise in proportion to the transdermal TRT dose and stay within normal range (typically with 25-100mg gel in men or 5-10mg gel or cream in women). Importantly, clinical effects are achieved only when serum testosterone levels are raised to the physiological range and are absent when serum levels fail to increase adequately. Using bone health as an example, studies show that serum testosterone levels above 200 ng/dl are required to prevent decreases in bone mineral density in men, which is usually achieved with 50-100mg of testosterone gel. This clinical correlation is consistently observed across many endpoints, including not only for bone mineral density, but also lean body mass, sexual function, hematologic parameters (erythrocytosis), endocrine feedback (luteinizing hormone suppression), and androgen-excess symptoms. 

Saliva testing produces supraphysiological results with transdermal TRT

Despite the establishment of serum as the evidence-based standard, many integrative/functional medicine providers are still taught to use saliva testing to monitor transdermal TRT. While endogenous saliva testosterone levels at baseline (no therapy) are usually consistent with corresponding serum measures of testosterone, this consistency is no longer observed once exogenous transdermal testosterone is introduced. The same standard doses of transdermal TRT that restore normal serum testosterone levels in proportion to the dose result in extremely elevated saliva testosterone levels, exceeding the physiological range and leading to clinical conclusions that the TRT dose is too high when it is in fact within the dose range demonstrated to be clinically effective. 

Elevated saliva responses to transdermal TRT lack clinical correlation 

Although the existence of a saliva–serum discordance is known and accepted among many integrative/functional medicine providers, there is little consensus surrounding its clinical interpretation. In one common interpretation, providers assume saliva levels reflect extensive absorption and transport of hormone to tissues, and serum levels underestimate tissue hormone exposure. However, this interpretation is not supported by the literature. We did not come across any published evidence showing that the elevated saliva responses to transdermal TRT are a reflection of clinical outcomes (while there is abundant evidence for serum, as discussed above).

In addition to reviewing the existing evidence, the article also presents new data exploring whether a clinical correlation for saliva testing might exist. In hundreds of women on transdermal TRT, saliva testosterone results were analyzed along with six androgen-excess symptoms, including excess facial or body hair, acne, irritability, scalp hair loss, low sex drive, and oily skin. As saliva testosterone levels increased to above-range levels in women on transdermal TRT, there was no corresponding increase in average symptom severity, nor was there a correlation between saliva testosterone levels and any of the six symptoms. These new data showing a lack of clinical correlation, together with the previous evidence of supraphysiological saliva responses to transdermal TRT, reinforce that what is occurring with testosterone levels in saliva may not be related to what is occurring systemically with testosterone at the tissue level. The collective evidence rules out saliva testosterone testing as a possible surrogate for serum testing during transdermal TRT monitoring.    

Saliva testing can lead to underdosing transdermal TRT

Relying on supraphysiological saliva results to adjust transdermal TRT doses often leads to selection of substantially lower doses of hormone therapy than those shown to have clinical efficacy. Studies suggest that serum testosterone levels must be above a certain threshold in order to provide protection for bone mineral density and muscle mass. If providers titrate TRT doses downward based on erroneously high saliva testosterone levels, patients may miss out on the full therapeutic benefits of TRT, not only for improvements in libido and energy, but also critically for bone and muscle preservation. Given the well-established relationship between testosterone deficiency and risks such as sarcopenia and low bone mineral density, underdosing TRT represents a meaningful clinical concern.

Saliva testosterone testing — Not validated for transdermal TRT monitoring but may be useful in other contexts 

Unlike on-therapy levels, endogenous (off-therapy) saliva levels of testosterone generally do correlate with those measured in serum, at least when using accurate saliva steroid assays (LC-MS/MS versus immunoassay), suggesting that saliva could be used for baseline measurement of testosterone. Saliva testing is also useful for detecting testosterone misuse, for example, in athletes. Essentially, saliva testing can answer the question, “Did this person use transdermal testosterone?”. This differs from the question, “Is this TRT dose effective?”, which is better answered with serum testing because of its alignment with androgenic clinical impact. 

Other serum alternatives (DBS and urine): Can they be used for TRT monitoring?

Dried bloodspot shows similar limitations as saliva

Testosterone levels in capillary DBS have generally been shown to correlate with serum testosterone (free and total) levels in individuals not on therapy, suggesting that DBS testing could be used to assess endogenous testosterone levels at baseline. However, like saliva, DBS often shows supraphysiological responses to transdermal TRT, and these responses are not known to be consistent with any clinical outcomes such as body composition or sexual function. Although DBS testing offers practical advantages over serum collection, the available evidence at this time is not sufficient to support the routine clinical use of DBS for monitoring transdermal TRT. 

Urine testing offers complementary value 

Androgen metabolites measured in urine have been shown to be highly correlated to serum testosterone (free and total) levels at baseline. After transdermal testosterone use, the rise in urine testosterone correlates to the rise in serum testosterone. As a caveat, urine testosterone does not reliably correlate to serum values in people with a certain gene variant (UGT2B17 deletion polymorphism), which leads to falsely low levels of testosterone in urine. Studies show that testosterone metabolites influence the response to TRT and its effects on physiologic outcomes in hypogonadal men. Although serum testing is better suited than urine for assessing testosterone deficiency before initiating TRT, urine androgen testing may be a useful complement to serum testing for transdermal TRT monitoring, to obtain additional non-overlapping information on hormone metabolism relevant for TRT outcomes.  

Takeaways for clinical practice 

For patients on transdermal TRT, the current evidence supports several key conclusions:

• Serum testosterone is the most reliable marker for guiding transdermal TRT dosing.
• Saliva testing lacks any evidence-based rationale for use as a surrogate for serum during transdermal TRT monitoring. 
• Evidence is insufficient to support the routine use of DBS for monitoring transdermal TRT.
• Urine testing may serve as an adjunct to serum during transdermal TRT monitoring, to provide complementary information about androgen metabolism. 

The article’s key findings prompt providers to carefully consider the clinical justification when selecting a testing method to monitor transdermal TRT, given the significant ramifications for patients. 

Read the full study here.

Reference

Newman MS, Smeaton J, Gillson G, Jaferi A (2026). Alternatives to serum testing for transdermal testosterone monitoring: A review of clinical data and correlation with clinical response. Frontiers in Reproductive Health 8:1804311.