Abstract: OBJECTIVE To establish an LC-MS/MS method for simultaneous determination of 15 bile acids in rats' serum. METHODS Serum samples were protein precipitated with acetonitrile and the separation was performed on a Xterra RP₁₈ column(4.6 mm×100 mm, 3.5 μm). All analytes were gradient eluted with acetonitrile and 10 mmol·L^-1 ammonium acetate in aqueous solution, of which flow rate was 1 mL·min^-1. Quantitative analysis of 15 bile acids was conducted by a triple-quadrupole mass spectrometer with negative-electrospray ionization(ESI) source, monitored under a multiple reaction monitoring(MRM) mode. RESULTS The calibration curves of 15 biles acids were linear in the quantitative range. The RSDs of the intra-day and inter-day precision results were all <10%. The average extraction recoveries of the control samples at low, middle and high concentrations were between 69.93%-88.83%. CONCLUSION Established LC-MS/MS method is simple, rapid and specific, and can be used for the determination of 15 bile acids in rat serum.