Which testosterone laboratory test should be ordered for women?

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Last updated: September 21, 2026 • View editorial policy

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Which Testosterone Test to Order for Women

Measure total testosterone using liquid chromatography-tandem mass spectrometry (LC-MS/MS) as the first-line test, and if total testosterone is normal but clinical suspicion remains high, add free testosterone (calculated from total testosterone and SHBG) or free androgen index (FAI). 1, 2

Primary Recommendation: Total Testosterone by Mass Spectrometry

The Endocrine Society guidelines clearly identify total testosterone as the key androgen to measure in women, but with a critical caveat: the assay method matters enormously 1. Standard automated immunoassays available in most hospital laboratories are not suitable for accurately measuring testosterone in women due to insufficient sensitivity and specificity at the low concentrations found in females 1, 3.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the gold standard method and should be used whenever available 1, 2, 4. If LC-MS/MS is unavailable, use a specialty laboratory with validated assays rather than routine hospital immunoassays 1.

When to Add Free Testosterone

Free testosterone is more sensitive than total testosterone for detecting androgen excess because it can be elevated even when total testosterone is normal 1. This occurs because women with hyperandrogenism commonly have low SHBG levels, which increases the free fraction 1.

Add free testosterone assessment when:

  • Total testosterone is normal but clinical features suggest hyperandrogenism (hirsutism, acne)
  • Risk factors for androgen excess are present (obesity, insulin resistance, family history of diabetes)
  • Hirsutism progresses despite therapy 1

How to Measure Free Testosterone

Do not use direct immunoassays for free testosterone—they are extraordinarily inaccurate 3. Instead, use one of these validated approaches:

  1. Calculated free testosterone (preferred): Compute from total testosterone, SHBG, and albumin using the law of mass action 3, 5
  2. Free androgen index (FAI): Calculate as total testosterone/SHBG ratio—reasonable correlation with free testosterone and useful when mass spectrometry is unavailable 2, 5
  3. Equilibrium dialysis or ultrafiltration: Reference methods but technically difficult and not practical for routine use 3

The 2025 international PCOS guidelines meta-analysis found that for diagnosing biochemical hyperandrogenism, total testosterone and free testosterone (by calculation or FAI) had the best diagnostic accuracy with pooled sensitivity of 0.74 and specificity of 0.86 for total testosterone 2.

Timing and Collection Details

Obtain blood samples:

  • Early morning (testosterone peaks in early morning) 1
  • Follicular phase (days 4-10) of menstrual cycle in regularly cycling women 1
  • Fasting sample for optimal accuracy 4

Note that testosterone varies by approximately 25% around the mean during the cycle, with slight increases at midcycle 1, 6.

Common Pitfalls to Avoid

  1. Using routine hospital immunoassays: These have systematic differences between assays and excessively broad normal ranges that miss androgen excess 1

  2. Relying on direct free testosterone immunoassays: These generate extraordinarily inaccurate values despite being simple and inexpensive 3

  3. Ignoring SHBG status: Women with obesity, metabolic syndrome, insulin resistance, or hypothyroidism have suppressed SHBG, which increases free testosterone even when total testosterone appears normal 1

  4. Measuring other androgens routinely: DHEAS, androstenedione, and DHT have poorer specificity and add little diagnostic value in most cases 1, 2. DHEAS is elevated in only 16% of women with normal total and free testosterone 1

Additional Considerations

If total and free testosterone are both normal but clinical suspicion for specific conditions remains:

  • 17-hydroxyprogesterone (early morning, follicular phase) in high-risk ethnic groups for nonclassic congenital adrenal hyperplasia: Ashkenazi Jews (1 in 27), Hispanics (1 in 40), Slavics (1 in 50) 1
  • Androstenedione and DHEAS may be considered if initial tests are normal, though they have poorer specificity 2

The key is recognizing that free testosterone correlates better with the androgenic state than total testosterone alone 3, but accurate measurement requires either calculation from validated total testosterone and SHBG assays or use of reference methods—never direct immunoassays.

References

3
Guideline

androgen therapy in women: an endocrine society clinical practice guideline.

The Journal of clinical endocrinology and metabolism, 2006

5
Research

Is it necessary to measure free testosterone to assess hyperandrogenemia in women? The role of calculated free and bioavailable testosterone.

Experimental and clinical endocrinology & diabetes : official journal, German Society of Endocrinology [and] German Diabetes Association, 2006

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This information is intended for healthcare professionals. Any medical decision-making should rely on clinical judgment and independently verified information. The content provided herein does not replace professional discretion and should be considered supplementary to established clinical guidelines. Healthcare providers should verify all information against primary literature and current practice standards before application in patient care. Dr.Oracle assumes no liability for clinical decisions based on this content.