A simple, sensitive and precise method for serum ferroxidase assay was developed by determining Fe (III) produced enzymatically from Fe (II) added as a substrate in an acetate buffer (pH 6.0). The Fe (III) formed was determined by flow injection analysis (FIA) using thiocyanate as a detection reagent. The reproducibility of this method throughout all steps was satisfactory (coefficient of variation, C.V.=3.4%), and the normal value of human serum ferroxidase activity was 0.30±0.02μmol Fe (III)/min/ml (mean±standard deviation). This method was applied to study the behavior of human and rabbit serum ferroxidases on Sephadex G-150 column chromatography.