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An analytical method based on hydrophilic interaction chromatography (HILIC) using diode array detection was developed and validated for the determination of epirubicin in human plasma. Epirubicin and epidaunorubicin (internal standard) were collected from the plasma by solid-phase extraction (SPE) using Oasis HLB cartridges. The chromatographic separation was on a silica analytical column (250 x 4.6 mm I.D., 5 mu m) with a mobile phase consisting of acetonitrile/40 mM ammonium formate buffer solution (pH 2.9) (90/10, v/v). The detection wavelength was 254 mn. Linearity of the method was in the concentration range of 0.0502 2.5 mu g/mL (r(2) = 0.9991). The limit of quantification (LOQ) and limit of detection (LOD) were 0.050 and 0.030 mu g/mL using 0.2 mL plasma sample, respectively. Recoveries of the analyte ranged from 89.4% to 101.2% with intra- and inter-day precision (RSD); less than 7.0% were obtained at the concentrations above the LOQ. The proposed method is simple, accurate, and its mobile phase is compatible for mass spectrometry.