A hydrophilic interaction chromatography-electrospray ionization tandem mass spectrometric method was developed for determination of anastrozole in human plasma. Anastrozole and irbesartan (internal standard) were extracted from human plasma with a mixture of dichloromethane and methyl tert-butyl ether (30:70, v/v). Analysis of the analytes was performed on a Luna HILIC column with the mobile phase of acetonitrile-10 m m ammonium formate (95:5, v/v) and detected by electrospray ionization tandem mass spectrometry in the selected reaction monitoring mode. The standard curve was linear (r(2)  = 0.9992) over the concentration range of 0.10-50.0 ng/mL using 200 μL of plasma sample. The coefficient of variation and relative error for intra- and inter-assay at four QC levels were 1.2-10.0% and -7.2-3.2%, respectively. The present method was applied successfully to the pharmacokinetic study of anastrozole after oral administration of 1 mg anastrozole tablet to healthy male volunteers.