Abstract: OBJECTIVE To develop an HPLC method for the determination of methadone hydrochloride in plasma. METHODS Agilent ZORBAX SB-C₁₈(4.6 mm×250 mm, 5 μm) column was used. The mobile phase was used acetonitrile- phosphate(pH 2.5)=32∶68. The flow rate was 1.5 mL·min^-1 and the UV detection wavelength was 206 nm. The internal standard was trihexyphenidyl hydrochloride. The plasma sample was extracted with ether, the organic phase was then extracted twice with 100 μL hydrochloric acid, took 20 μL sample from water-phase for analysis. RESULTS The concentration of methadone hydrochloride had a good linear relationshipin within 0.1-10 mg·L^-1(r=0.999 6); the lower limit of quantification was 0.05 mg·L^-1; the mean relative recovery was (96.89±0.64)%; the average absolute recovery was (77.68±2.47)%. The within-day and the between-day RSD were less than 5%. CONCLUSION The method is simple, rapid, accurate, and can be used for drug determination of methadone hydrochloride in plasma.