OBJECTIVE To develop a simple reversed-phase high-performance liquid chromatography(HPLC) method for thesimultaneous determination of the antiepileptic drugs(AEDs) phenytoin(PHT),its prodrug fosphenytoin(FOS),and its metabolite 4′-hydroxyphenytoin(4′-HPPH) in human plasma.METHODS Plasma sample(100 μL) pre-treatment involved acidification with 20% phosphoric acid,simple extraction with 1 mL of acetic ester containing 10 mg·L^-1 propranolol hydrochloride as internal standard and evaporation of solvent,followed by injection of reconstituted sample onto a Agilent RX-C₈ column(4.6 mm × 250 mm,5 μm).The mobile phase consisted of methanol-acetonitrile-0.1% triflouroacetic acid(23∶17∶60),and delivered at a 1.5 mL·min^-1.The analytes were detected at the wavelength of 210 nm.RESULTS The method was found to be linear over the concentration ranges investigated,1-400 mg·L^-1 for FOS,0.5-50 mg·L^-1 for PHT and 0.5-10 mg·L^-1 for 4′-HPPH,respectively.The intra-and inter-day reproducibility was good with the RSD of 8% or below and relative recovery was ranged from 92.79% to 107.90% for all analytes.CONCLUSION This method was proved to be accurate,sensitive and convenient and suitable for clinical therapeutic drug monitoring.