This paper describes high performance liquid chromatography (HPLC) conditions suitable for separating hydroperoxy-phospholipids from hydroxy-phospholipids. 1-stearoyl-2-(13-hydroperoxy-cis-9,trans-11-octadecadienoyl)-L-3-phosphatidyl-choline, 1, 2 di (13-hydroperoxy-cis-9,trans-11-octadecadienoyl)-L-3-phosphatidylcholine, 1-palmitoyl-2-(13-hydroperoxy-cis-9,trans-11-octadecadienoyl)-L-3-phosphatidylethan-olamine and their corresponding hydroxy-phospholipid derivatives were resolved by HPLC on an Ultracarb 5 ODS (20) column at 30C. Each pair of these phospholipids required different conditions for optimal separation but all used a mobile phase of acetonitrile-methanol-water mixture containing 10 mM choline chloride. The separation described allows accurate measurement of phospholipid hydroperoxide peroxidase activities.