Lysophosphatidylcholine (LPC) is a bioactive lipid implicated to play a functional role in various diseases including atherosclerosis, diabetes, cancer, and inflammation. Conventional methods are of limited value for a systematic evaluation of LPC species concentrations due to complicated, time-consuming procedures. Here we describe a methodology based on electrospray ionization tandem mass spectrometry (ESI-MS/MS) applicable for high-throughput LPC species quantification. This assay provides accuracy and precision sufficient for the analysis of large clinical studies as well as basic biochemical studies in a broad range of biological material including plasma, tissues, and cell culture material. This method may be combined with methods based on the same analytical setup for glycerophospholipid, sphingolipid, and sterol analysis to evaluate LPC species in relation to other lipid species profiles.