Objective To determine whether liquid chromatography-tandem mass spectrometry (LC-MS/MS) determination of total (TT) and free (FT) testosterone is more specific than extraction chromatography-radioimmunoassay (RIA) for distinguishing women with polycystic ovary syndrome (PCOS) from controls and whether differing cutoff values should be used depending on the setting. Design Prospective case-control cohort study. Setting Tertiary care center and reference laboratory. Intervention(s) Blood sampling. Patient(s) One hundred patients with PCOS and 100 controls. Main Outcome Measure(s) Circulating TT measured by RIA and LC-MS/MS and FT measured by equilibrium dialysis using RIA or LC-MS/MS-generated TT values. Result(s) T measurement by RIA and LC-MS/MS similarly differentiated patients with PCOS from controls; detection of PCOS was higher for FT for both methods. TT values demonstrated greater overlap between patients with PCOS and controls than did FT for both RIA (80% vs. 42% overlap) and LC-MS/MS (52% vs. 67% overlap). A lower cutoff value by LC-MS/MS was better suited for the study of patients seen in the clinical (referred) setting (35 and 4.0 ng/dL for TT and FT, respectively) than in the screening of a general population (50 and 5.0 ng/dL for TT and FT, respectively). Conclusion(s) Extraction chromatography-RIA and LC-MS/MS measurements of T have similar performance for differentiating patients with PCOS from healthy controls; LC-MS/MS may be preferable given its relative ease of automation. Compared with FT, measurement of TT has relatively limited value for distinguishing PCOS from normal. Finally, different cutoff values should be considered depending on the clinical/investigative setting, with higher values being used in the study of biased (e.g., clinical or referred) populations. © 2014 American Society for Reproductive Medicine, Published by Elsevier Inc. All rights reserved.