Objectives: Ethanolamine ether phospholipids (ePE) and choline ether phospholipid (ePC) are present in human serum or plasma. Decreases in ether phospholipids (plasmalogens) in serum (plasma) have been reported in several diseases such as Alzheimer's disease, Parkinson's disease, metabolic syndrome, schizophrenia. Therefore, need for assay of ether phospholipids in plasma may increase in the future. Nowadays, measurement of the ether phospholipids in human plasma seem to depend on tandem mass spectrometry (LC/MS/MS), but a system for LC/MS/MS is too expensive for most of ordinary clinical laboratories, moreover, use and maintenance of the system are time consuming. Design and methods: Phospholipase A1 (PLA1) hydrolyzes ester (acyl) bond at the sn-1 position of glycerophospholipids, but it does not act on ether bond at the sn-1 position. We confirmed by a HPLC method that treatment of plasma with PLA1 causes complete disappearance of all diacyl phospholipids, but ether phospholipids remain intact. On the basis of these observations, we developed an enzymatic assay method for ePE and ePC in human plasma by use of a fluorescence plate reader. Results: The amount of ePE in human plasma measured by the enzymatic method was well correlated to that by LC/ESI-MS method (R2 > 0.94), but the correlation of ePC between the two methods was bit poorer (R2 > 0.77) than that of ePE. Conclusion: The enzymatic method may be applied to assay of ether phospholipids (ePE and ePC) not only in human plasma but also to assay of ePE and ePC in the other tissues.