Abstract: OBJECTIVE To develop an ultra performance liquid chromatography-tandem mass spectrometry method for the determination of barlerin in rat plasma. METHODS Plasmas were precipitated with 200 μL acetonitrile. An ACQUITY UPLC BEH C₁₈(50 mm×2.1 mm, 1.7 μm) column was used as the stationary phase. The mobile phase was consisted of acetonitrile and 0.1% formic acid water with gradient elution pumped at a flow rate of 0.4 mL·min^-1. The analytes were detected with positive electrospray ionization in multiple reaction monitoring(MRM) mode and quercetin was used as internal standard. RESULTS Excellent linear calibration curve of barlerin was obtained in the concentration range of 2.5-500 ng·mL^-1(r=0.997 9). The lower limit of quantification were 0.5 ng·mL^-1. The intra and inter day relative standard deviations of low, medium and high quality control samples (3, 45 and 450 ng·mL^-1) were all <10%. The average recovery were (103.59±4.75)%, (98.68±4.62)% and (97.06±5.64)%. CONCLUSION The method is simple, rapid and sensitive, which is suitable for pharmacokinetics study of barlerin in rats.