Abstract: Using high performance liquid chromatography (HPLC) and spectrophotometry (UV) for determining the concentration of phenytoin in rabbit plasma, both methods show linearity of standard curve. The overall recoveries of drug by HPLC and UV are 99.14±1.70% and 101.50±2.28%, respectively. The results of the determination of the concentration of phenytoin in plasma by HPLC and UV after intervenous injection of single dose of 10 mg/kg in rabbits were similar, exhibiting a good agreement between these two methods. HPLC has the following advantages: (1) it is a specific, sensititive and reliable micro-method; (2)it does not necessitate derivatization of the compound; (3) it only requires a relatively small sample as low as 50～100μl; (4) it can be used for simultaneous determination of several drugs in plasma.