Simple and rapid quantitative analysis utilizing high-performance liquid chromatography was performed to determine the concentration of the antiepileptic agent phenytoin, and its main metabolite hydroxyphenytoin, in human urine. For the purposes of simple and rapid determination, the commercially available Extrelut columns were used with diethyl ether and chloroform as extraction solvents. High-performance liquid chromatography was performed on a LiChrosorb RP-8 column, with a mobile phase of methanol-0.02% ammonium acetate (1:1). The internal standard was 5-(4-methylphenyl)-5-phenylhydantoin. The method was applied to patients' urine, to examine the influence of concomitant drugs. Also, the results obtained using the commercially available enzymatic immunoassay method were compared with those from the present method, and it was concluded that a simple and rapid microanalysis is possible with a high extraction ratio.