Abstract A high-performance liquid chromatographic method for the quantification of major phospholipid classes is described. The separation was performed on Ultrasphere SI silica gel columns with a mobile phase of acetonitrile—methanol—85% phosphoric acid (100:10:1.8, v/v) using isocratic elution and UV detection at 203 nm. Complete separation of phosphatidylserine, phosphatidylethanolamine, plasmalogen, phosphatidylcholine and sphingomyelin was achieved within 8 min. The plasmalogen was resolved from phosphatidylethanolamine in hydrochloric acid-derivatized samples, or without derivatization using a mobile phase composition of 100:40:0.4. The phospholipids were quantified by peak-area integration by means of the calibration. The detection limit is 5 ng. Human erythrocyte ghost membranes, lymphocytes and thrombocytes were analysed for these phospholipids. This method is suitable for routine clinical studies of membrane disorders in health, toxicity and disease, as well as in research.