Abstract: In this study 2-hydroxyflutamide extracted from human plasma with dichloromethane and determined by HPLC using UV detection. The chromatographic system consisted of Nova-Pak CN cartridge, an isocratic mobile phase of acetonitrile-tetrahydrofuran-water (31.5: 2.5: 66, v/v/v) and UV detection at 300 nm. 2-Hydroxyflutamide and internal standard were eluted at about 4.2 and 5.8 min, respectively. The method was linear over the range of 10-1000 ng mL-1 of 2-hydroxyflutamide in plasma (r2>0.9998). The within-day and between-day precision values were in the range of 1.96-6.06%. The limit of quantification of the method was 10 ng mL-1. The method was successfully applied for the study of the pharmacokinetics of flutamide in healthy volunteers.