Background:To assess the analytical validity of free testosterone (FTe) measurements, a reference measurement procedure (RMP) is required. For steroids, isotope dilution-mass spectrometry is accepted as state-of-theart technology. Because FTe is defined as the hormone fraction in serum water in equilibrium with the proteinbound fraction, the RMP should include a physical separation step. The use of equilibrium dialysis (ED) or ultrafiltration (UF) is advocated. Our objective was to develop such a candidate RMP. Methods: We selected UF combined with isotope dilution-gas chromatography-mass spectrometry (ID-GC/ MS) for direct measurement of Te in the ultrafiltrate. After optimization of the UF process, the complete procedure was validated by use of split-sample comparisons with indirect ED (iED) and symmetric dialysis (SyD). Results: The candidate RMP gave maximum withinday, between-day, and total CVs of 3.0%, 3.1%, and 4.3%