The levels of ursodeoxycholic acid and chenodeoxycholic acid in rat plasma were determined by LC-MS. The effects of ursodeoxycholic acid and chenodeoxycholic acid Pharmacokinetic behavior in mice. Methods: The plasma samples were treated with methanol, ethyl acetate and formic acid (50:50: 1) mixed solvent. Diclofenac was used as internal standard. The column was a Waters Atlantis T3 C18 column (150 mm × 2.0 mm, 5 μm) Water (containing 1 mmol·L-1 ammonium acetate and 0.01% formic acid) -methanol (76:24), isocratic elution at a flow rate of 0.2 m L · min-1, Shimadzu LC-MS2020 MS single quadrupole Device, negative ion mode. Rats were fed with 100 mg · kg-1 bolus extract, and the plasma concentration of ursodeoxycholic acid and chenodeoxycholic acid was determined by LC-MS. Pharmacokinetic parameters were calculated by DAS 2.0 software. Results: The results showed that the endogenous impurity did not interfere with the determination of ursodeoxycholic acid, deoxycholic acid and internal standard, the linear range was 0.078 ~ 10.0μg · m L-1, the limit of quantification was 0.078μg · m L-1. The precision, accuracy, stability and recovery rate of the method are in accordance with the requirements of biological samples. It is suitable for the determination of two kinds of bile acids in rat plasma. The method can be applied to the treatment of ursodeoxycholic acid and chenodeoxycholic acid Pharmacokinetics of Drugs. Conclusion: The method is simple, sensitive and specific. The methodological research is in accordance with the requirements of biological samples and is successfully used in the study of pharmacokinetics of ursodeoxycholic acid and chenodeoxycholic acid in rats.