Concentration of both choline plasmalogen (PC-Pls) and ethanolamine Pls (PE-Pls) in human plasma/serum has been getting attention to, since certain patients including those with neurodegenerative disorders, have been reported to exhibit reduced levels of specific Pls species. However, despite using liquid chromatography–tandem mass spectrometry (LC–MS/MS), accurate quantitation of Pls is still difficult because of less product ion from PC-Pls and quantitative issues (e.g., extraction recoveries and matrix effects). The present study aimed to develop a method for accurate identification and quantitation of Pls molecular species using LC–MS/MS operated in the multiple reaction monitoring mode. The LC–MS/MS conditions in the presence of sodium, and the extraction method using methanol protein precipitation were optimized. Under the optimal condition, Pls was detected at femtomole levels. The recoveries of Pls from human plasma were nearly 100%, and matrix effects were not observed. The novel method enabled determination of each Pls species in human plasma at the concentrations of 0.5–13.6 μM. Then the PC-Pls and PE-Pls species in the plasma of both healthy subjects and patients with Alzheimer's disease were quantitated. The method developed herein represents a powerful tool for analyzing Pls, which may provide a better understanding of their physiological roles in vivo.