A relatively little time consuming simple method based on the treatment of bile with cholesterol oxidase and subsequent high performance liquid chromatography measurement of the 3-ketocholesterol produced in order to determine the level of the cholesterol concentration is described. The method avoids bilirubin interferences, has high reproducibility and recovery assays give 100 % values. It is highly sensiti\·e and suitable for use in the determination of cholesterol concentrations in bile and other bilirubin containing biological fluids.