〔Abstract〕 Objective High performance liquid chromatography-mass spectrometry(LC-MS/MS) system was used to accurately determine 22 bile acids in serum, liver, amniotic fluid and placenta of pregnant mice, and a LC-MS/MS method was established for efficient detection and analysis of bile acids in serum, liver, amniotic fluid and placenta of mice. Methods Pregnant mice serum, liver, amniotic fluid and placenta samples were processed, with 0.1% glacial acetic acid in 4 mmol/L ammonium acetate aqueous solution as mobile phase A and pure methanol as mobile phase B, the flow rate was 0.4 ml/min, a gradient elution program was used to elute with Phenomenex Gemini 3 μm NX-C18 110A(100 mm × 2.0 mm) chromatographic column elution, and mass spectrometry detection system used an electrospray ion source for negative ion multiple reaction monitoring. Results The linear relationship of 22 bile acids in the quantitative range was good. The RSD of inter-day and intra-day precision at low, medium and high concentrations was 0.5%-7.4%, the matrix effect was 88%-110%, and the extraction recovery was 84%-108%. Conclusion In this experiment, LC-MS/MS was established to detect 22 bile acids in serum, liver, amniotic fluid and placenta of pregnant mice. The method not only has high sensitivity and selectivity, but also can stably detect a large number of samples.