Abstract: OBJECTIVE To establish a LC-MS/MS method for the simultaneous determination of rutin and hydrochlorothiazide in rat plasma and to study the pharmacokinetics of Zhenju Jiangya Pill in rats. METHODS The plasma sample was handled by protein precipitation. The column was Pntulips BP-C₁₈ column(2.1 mm×50 mm, 5 μm) with the mobile phase of acetonitrile and water by gradient elution at 0.45 mL·min^-1. The column temperature was 30℃. The electrospray ion source and the ion reaction monitoring mode were used. DAS 2.0 software was used to calculate the pharmacokinetic parameters. RESULTS Methodological verification results showed that the endogenous impurities did not interfere with the determination of the analytes and internal standard. The linear ranges of rutin and hydrochlorothiazide were 5-1 000 ng·mL^-1(r²=0.997 1) and 2.5-500 ng·mL^-1(r²=0.995 8), respectively. The pharmacokinetic parameters of rutin and hydrochlorothiazide were as follows:AUC_(0-t) were (107 157.31±38 056.63), (130 387.28±46 306.69)ng·mL^-1·min, T_1/2z were (108.65±20.95), (240.86±46.44)min, T_max were (34.25±16.34), (120.00±0.00)min, C_max were (683.44±254.03), (368.45±136.95)ng·mL^-1. CONCLUSION The precision, accuracy, mean recoveries and the matrix effects were satisfied with the requirements of biological sample measurement. The method was successfully applied to the pharmacokinetic study of Zhenju Jiangya Pill in rats.