The methods for detecting endogenous phospholipids (PL) are focused on the use of liquid chromatography tandem mass spectrometry (LC-MS/MS). Since the structural relation and similar core structure of glycerophospholipids results in a common and predictable mass spectrometric fragmentation behavior, in the present study, six classes of lysoglycerophospholipid (Lyso-PL) were subjected to a predict-verify approach by multiple reaction monitoring (MRM) mode based on liquid chromatography-quadrupole linear ion trap mass spectrometry (LC-QTRAP-MS/MS) complemented with liquid chromatography-quadrupole time of flight mass spectrometry (LC-QTOF-MS/MS) in biological serum samples. A targeted metabolite profile of Lyso-PL was established and represented in the form of a LC-QTRAP-MRM analysis. There were total of 96 Lyso-PL molecular species, consisting of 317 regioisomers detected in serum of human and four rodent species. In addition, the metabolite profile was successfully applied to the analysis of qualitative distribution of Lyso-PLs in serum of human and four rodent species. We believe that this improvement in the method for qualitative analysis of endogenous Lyso-PLs should greatly contribute to identifying the role of lipid molecular species in biological systems. Also, the integrated strategy for establishment of metabolite profile can be applied to targeted qualitative analysis of other endogenous metabolites which occur in a high individual number but sharing either structural similarities or similar functional groups.