Ebselen is an organo-selenium, highly hydrophobic compound that exhibits glutathion peroxidase-like activity. It is now in phase III trials in Japan for investigating its effect on ischemic stroke. We have developed an HPLC method for the determination of Ebselen at ambient temperature and applied it to a patented tablet formulation. The mobile phase composition, detection wavelength and chromatographic conditions were optimized. The optimum conditions are a Waters, C-18, (25 cm x 4.6 mm, 5 mum) column, 1% acetic acid: methanol: propanol (40:50: 10) (v/v) as the mobile phase at a flow rate of 0.7 mL min(-1) and detection at 254 nm (internal standard: benzanilide). The method was validated and calibration curves were constructed depended on the ratios of Ebselen to benzanilide peak areas.