Objective: The objective of our study was to determine reference intervals and biologic variation for testosterone (T), free testosterone (fT), and free androgen index (FAI) in women with accurate methods and to test the discriminative value of these parameters in a polycystic ovary syndrome (PCOS)-population. Methods: Serumwas obtained daily during a normal menstrual cycle from 25 healthy women (677 data-points). A single serum sample was obtained from 44 PCOS-patients. T was measured by LC-MS/MS and by Architect® 2nd generation T Immunoassay. Sex hormone-binding globulin was measured to calculate fT and FAI. Results: Reference intervals which were established in healthy women with an ovulatory menstrual cycle were T = 0.3-1.6 nmol/L and 0.5-2.0 nmol/L, fT = 5.2-26 pmol/L and 7.2-33 pmol/L, and FAI = 0.4-2.9 and 0.6-4.4, by LC-MS/MSand immunoassay, respectively. T, fT and FAIwere higher in PCOS patients than in controls (p b 0.0001). The areas under the curve of receiver operator characteristic (ROC) plots were not different for T, fT, or FAIwhen Twasmeasured by LC-MS/MSversus immunoassay based on prediction of PCOS. FAI and fTwere the strongest predictors of PCOS. Conclusions: When based upon the appropriate reference intervals and ROC analysis, LC-MS/MS and second generation immunoassay have equivalent clinical utility for the diagnosis of PCOS.