Abstract: OBJECTIVE To propose a UPLC-MS/MS method for the determination of methadone hydrochloride and its metabolites 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine(EDDP) in rat plasma. METHODS Diazepam was used as internal standard, rat plasma was precipitated with acetonitrile, the concentration of methadone hydrochloride and EDDP in the plasma of rats after intragastric administration methadone 6 mg·kg^-1, determined by UPLC-MS/MS, and the data were analyzed. RESULTS The limit of quantitation was 0.5 ng·mL^-1 for both methadone hydrochloride and EDDP in rat plasma; the linear correlation coefficient r>0.999; the intra-day and inter-day RSDs were all < 15%; Absolute recovery rates were above 85%. The relative recoveries ranged from 90% to 110%; the matrix effect ranged from 92.80% to 97.71%. The main pharmacokinetic parameters of methadone and EDDP were as follow:t_1/2 of methadone and EDDP were (1.479±0.839), (2.590±0.760) h; C_max were (48.562±10.501), (25.542±3.706) μg·L^-1; MRT_0-t were (3.476±0.310), (3.883±0.514) h; AUC_0-t were (204.036±61.145), (86.031±15.287) μg·h·L^-1; CL were (32.338±14.343), (70.137±10.786) L·h^-1·kg^-1, respectively, and there were significant differences in the parameters between the rats before and after being administrated with methadone and EDDP. CONCLUSION The method is proved to be highly sensitive, selective, and has been successfully applied to the determination of methadone hydrochloride and its metabolite EDDP, and can be used for pharmacokinetic studies in rats.