In this study, we developed and validated a High-Performance Liquid Chromatography-Tandem Mass Spectrometry (HPLC-MS/MS) method to determine bile acid in mouse serum. The serum samples were analysed after solid-phase extraction. The analytes were separated on a Diamonsil C18 column with a mobile phase of methanol and water containing 10 mmol/L ammonium acetate and 0.005% formic acid (70:30) at a flow rate of 0.5 mL/min. Analytes were detected by tandem mass spectrometry in negative ion mode. The results demonstrated that the calibration curve was linear for all bile acids over a range of 10-10000 ng/L. The specificity, matrix effect, recovery, linearity, accuracy, and precision were validated for bile acid in mouse serum. The HPLC/MS/MS method was selective, sensitive, and simple, and was applied successfully to determine the bile acid in more than 200 mouse serum samples. In conclusion, this method is suitable for the quantitative detection of bile acid.