Abstract: A high-performance liquid chromatography tandem mass spectrometry(LC-MS/MS)method was developed for the simultaneous determination of bile acids, bilirubin and taurine in rat plasma. Plasma samples were protein precipitated with methanol and analyzed by LC-MS/MS. The analytes were well separated on a Zorbax SB-C₁₈ column(150 mm×2. 1 mm, 5 μm)with gradient elution of methanol and 10 mmol/L ammonium acetate in aqueous solution(pH 3. 0). Detection was carried out on a triple quadrupole tandem mass spectrometer by selected reaction monitoring(SRM)in the negative ion mode. The proposed method proved to be sensitive and selective, which is suitable for the determination of bile acids, bilirubin and taurine in rat plasma.