Background: Quantification of bioactive, free serum cortisol concentrations can characterize adrenocortical function more appropriately compared to total serum cortisol measurement. Ultrafiltration or equilibrium dialysis of serum samples allow direct measurement of free serum cortisol concentrations but respective methods have poorly been validated so far. The aim of our study was to investigate the analytical performance of free serum cortisol measurement employing equilibrium dialysis and ultrafiltration. Methods: Two commercially available ultrafiltration devices and self-assembled dialysis cells, respectively, were studied. Cortisol was quantified in filtrate or dialysate using an automated immunoassay system. Using two serum pools, the inter-assay coefficient of variation was determined for the three methods and a method comparison was performed. Results: Inter-assay coefficients of variation (n=10) between 3.2% and 14.8% were observed in the imprecision study. Method comparison demonstrated close agreement between free serum cortisol results obtained by ultrafiltration and equilibrium dialysis, respectively (equilibrium dialysis = 1.2x ultrafiltration+3.9 nmol/L; r=0.99; n=35). Conclusions: Direct quantification of free serum cortisol after equilibrium dialysis or ultrafiltration of the samples offers acceptable reproducibility and results in close agreement can be obtained. Both methods can potentially be introduced into a routine laboratory setting.