The levels of lysophosphatidic acid (LPA) or lysophosphatidylcholine (LPC) in plasma have been shown to be markers for several human diseases, including cancers. Here we show that the presence of LPC or other lysophospholipids (LPLs) in lipids extracted from biological samples affects accurate measurement of endogenous LPA in biological samples. We report for the first time the artificial conversion of LPC and lysophosphatidylserine (LPS) to LPA at the ion source of electrospray ionization tandem mass spectrometry (ESI-MS/MS). To avoid the interference of LPC with the quantification of LPA, a method based on high-performance liquid chromatography (HPLC) separation of LPA from LPC has been developed.