Background Bile acids, as important signaling molecules and regulatory factors acting on glucose, lipid, and energy metabolism, are always involved in liver, biliary, and intestinal diseases. Development and validation of a simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of bile acids is significant for the routine clinical testing. Methods Fifty microlitre of serum was mixed with 10 mu l of the internal standard working solution and then 140 mu l of methanol for protein precipitation. After centrifuged, the supernatant was directly used for LC-MS/MS analysis. Results Good separation of all bile acid species was achieved. The method was validated with consistent linearity for individual bile acids, good recovery, low carryover, satisfactory sample stability, and analytical specificity against hemolysis, lipemia, and bilirubinemia. The intra-day and the inter-day imprecision values were in the range of 1.53%-10.63% and 3.01%-13.98%, respectively. No obvious matrix effect was observed. The reference intervals of bile acids in adults have been established for the clinical testing. Conclusions The low sample volume, simple sample preparation, good separation of all species, and satisfying validation results make this LC-MS/MS approach suitable for usage as a high-throughput assay in routine clinical laboratories.