Background: Testosterone concentrations in normally cycling women are assumed to be elevated around the time of ovulation. The clinical relevance of changing testosterone concentrations during the menstrual cycle, however, is unclear. Poor performance of current direct immunoassays for testosterone at low concentrations confounds this issue. Therefore, our objective was to assess daily testosterone fluctuation during the menstrual cycle by a thoroughly validated isotope dilution-liquid chromatography-Tandem mass spectrometry (ID-LC-MS/MS) method and to evaluate whether an ARCHITECT® 2nd Generation Testosterone fully automated immunoassay is equally suited for this purpose. Methods: Testosterone was measured in serum obtained daily during the menstrual cycle of 25 healthy women, characterized by biochemical and physical examination. Results: Performance of the ID-LC-MS/MS method was concordant with a published reference method (y = 1.007x - 0.056 nmol/L; r = 0.9998). Comparison of the immunoassay to ID-LC-MS/MS yielded y = 1.095x + 0.104 nmol/L (r = 0.9031). Overall, testosterone concentrations were higher mid-cycle, but a peak was not discernible in each individual. Apart from a persistent positive bias, the immunoassay measured the same testosterone profiles as the ID-LC-MS/MS method. The reference interval in women was 0.30-1.69 nmol/L (8.7-48.7 ng/dL) for ID-LC-MS/MS and 0.50-2.00 nmol/L (14.4-57.7 ng/dL) for the immunoassay. Conclusion: The elevation of mid-cycle testosterone concentrations is statistically significant, although not clinically relevant since day-To-day variation is higher and independent of the menstrual cycle. In this light, a single testosterone measurement might not be reflective of the overall testosterone status in an individual. Measurements obtained using the 2nd generation immunoassay gave comparable results across the menstrual cycle. © 2012 Elsevier Inc. All rights reserved.