OBJECTIVE： To establish a method for simultaneous determination of methadone， venlafaxine and their metabolites in rat plasma， and to use it for the study of pharmacokinetic in rats. METHODS： UPLC-MS/MS method was adopted to determine plasma after precipitated with acetonitrile using diazepam as internal standard. The determination was performed on Waters Acquity UPLC BEH C18 column with mobile phase consisted of acetonitrile-0.1％ formic acid （gradient elution） at the flow rate of 0.4    mL/min. The column temperature was set at 40 ℃， and sample size was 2 μL. ESI was used for positive ion scanning by multiple reaction monitoring （MRM） mode. The ion pairs for quantitative analysis were m/z 310.4→265.4（methadone）， m/z 278.2→234.1（EDDP）， m/z 278.1→57.8（venlafaxine）， m/z 263.9→57.9（O-desvenlafaxine）， m/z 285.1→193.1（internal standard）. Eight SD rats were given methadone hydrochloride 6 mg/kg and venlafaxine hydrochloride 10 mg/kg intragastrically （4 rats for each drug）； blood samples （0.3 mL） were collected from the tail vein before and 0.083， 0.167， 0.25， 0.5， 0.75， 1， 1.5， 2， 3， 4， 6， 8， 10， 12， and 24 h after medication. Pharmacokinetic parameters were calculated by using DAS 3.0 software. RESULTS： The linear ranges of methadone， EDDP， venlafaxine and O-desvenlafaxine were 0.5-250 ng/mL （r＝0.999 7）， 0.5-250 ng/mL （r＝0.999 2）， 0.4-200 ng/mL （r＝0.999 9）， 0.4-200 ng/mL （r＝0.999 9）， respectively. The limits of quantitation were 0.5， 0.5， 0.4， 0.4 ng/mL. RSDs of intra-day and inter-day were all lower than 9.0％ （n＝6）. The method recoveries were 94.20％-102.87％， 90.93％- 102.94％， 92.95％-101.61％， 90.33％-101.97％（RSD≤5.5％，n＝6）. The extraction recoveries were 85.90％-94.45％， 85.97％- 91.66％， 87.97％-93.58％， 88.53％-94.54％（RSD≤5.9％，n＝6）. The matrix effects were 95.96％-97.78％， 92.33％-97.40％， 95.28％-97.71％， 95.33％-95.74％（RSD≤4.9％，n＝3）. The pharmacokinetic parameters included that t1/2 were （1.42±1.02）， （2.59±0.76），（0.63±0.08），（1.29±0.14） h；cmax were （52.21±5.42）， （25.68±3.45），（45.68±2.29），（47.63±13.09） μg/L； MRT0-24 h were （3.55±0.21）， （3.98±0.41），（1.44±0.21），（2.01±0.17） h；AUC0-24 h were （201.95±51.14）， （86.092±15.95）， （75.38±23.95），（82.90±23.44）μg·h/L. CONCLUSIONS： The method is specific， absolute separated， rapid and sensitive， and can be used for the simultaneous determination of methadone， venlafaxine and their metabolites in plasma and pharmacokinetics research.