Abstract A single high-performance liquid chromatographic method is described for determining and measuring the major conjugated bile acids in human bile. Four different chromatographic conditions were applied using C-18 columns and an isocratic solvent system. An effective one-step purification with Sep-pak C-18 was adopted. The advantages of this method are an improved separation of the conjugated bile acids within a short period of time. The detection limit was 0.0125 μg/μl and linearity was up to 6 μgg/μgl. Recovery was always up to 96.6 %, this being sufficient for routine clinical application during bile acids evaluation in biliary lithiasis.