Testosterone in serum may be unbound (free), or bound to either sex hormone binding globulin (SHBG) or albumin. Consequently, total serum testosterone analysis may be misleading in situations where binding protein concentrations are abnormal. Current methods for estimating the biologically active (bio-available) serum testosterone concentration involve physical separation of testosterone fractions and are not amenable to high-throughput analysis. The use of automated immunoassays for total serum testosterone has also been questioned. Liquid chromatographytandem mass spectrometric (LCMS/MS) methods for measuring testosterone have gained favour due to their superior selectivity. The separation of testosterone fractions by turbulent flow chromatography (TFC TurboFlow technology, ThermoFisher Scientific) prior to MS/MS analysis.was investigated. TFC is based on the direct injection of biological samples onto a column packed with relatively large particles at high flow-rates. In the resulting turbulent flow, smaller analytes can enter the column interstices but larger proteinaceous material is excluded. Serum from a male volunteer was analysed (i) following protein precipitation with equal volumes of methanol (total testosterone) and (ii) by direct injection onto the TFC column (retained fraction). By comparison of peak areas, it was found that 25% of the total serum testosterone was retained after direct injection. This agrees well with an ammonium sulfate precipitation method for bio-available testosterone (22%) used on the same sample. Based on the known binding affinities of SHBG and albumin for testosterone, it is likely that the more weakly bound albumin fraction was, to some degree, dissociated during the TFC process. Testosterone and SHBG were added separately to serum pools and analysed as previously described. The peak area ratio was independent of total testosterone concentration, but decreased at higher SHBG concentrations. Whilst further validation is required to prove the utility of TFC, it has considerable potential for the analysis of bio-available steroid hormones in serum.