Abstract: In this paper a comprehensive method for the determination of bile acidsin serum was described. Total serum bile acids were extracted and purified bySEP-PAK C18 Cartridge and were grouped with anion exchange resin PHP LH-20, conjugated bile acids were converted to free form by alkaline hydrolysisunder high pressure and temperature. A superior method for trimethylsilylationof bile acids is also described. The assay was based upon two deuterium label-led bile acid as internal standard and selected ion monitoring.