We have developed a method for assaying the activity of phospholipid hydroperoxide glutathione peroxidase (PHGPx) which is both more sensitive and specific than the spectrophotometric assay. The assay is based on the direct detection of the enzymatic product 1-palmitoyl-2-(13-hydroxy-cis-9, trans-11-octadecadienoyl)-L-3-phosphatidylcholine by HPLC. Under the conditions used, baseline separation is achieved for product and substrate. The utility of the method is demonstrated by the measurement of PHGPx activity in crude extracts from human lenses and from human Hep G2 hepatoma cells. This method is also suitable for measuring the specificity of PHGPx for cofactors apart from glutathione. The assay was used to demonstrate that cysteine alone at pH 7.4 mimics PHGPx activity.