Abstract: Utilizing the function of collision energy automatic optimization, the fragmentation patterns of the phospholipids standards, including classes of glycerophosphatidic acid (PA), glycerophosphocholine (PC), glycerophosphoethanolamine (PE), glycerophosphoserine (PS), glycerophosphoglycerol (PG) and glycerophosphoinositol (PI) were studied both in negative and positive modes by triple quadrupole tandem mass spectrometry. It could be inferred that, for all classes of phospholipids, the intensities of the ions arising from neutral loss of the sn-2 substituent as a free fatty acid (M-H-R₂COOH^-) or ketene (M-H-R`₂CH=CO^-) are stronger than those of corresponding ions generated by loss of the sn-1 substituent. According to this rule, it was readily to confirm the position of acyl group. In addition, it was easy to elimination of carboxylate substituent from sn-2 position for PC, which meant that the intensity of R₂COO^- was higher than that of R₁COO^-. While for PA and PS, the phenomena were totally opposite to that of PC and the intensity of R₂COO^- was weaker than that of R₁COO^-. When in the positive ion mode, the intensity of acylium ion from the sn-1 position was weaker than the acylium ion from the sn-2 position for PS. Moreover, at low collision energy, for PA and PC species it could be found that the intensities of the ions arising from neutral loss of the sn-2 substituent as a ketene (M-H-R’₂CH=CO^-) were greater than those of ions reflecting corresponding losses of the sn-1 substituent at low collision energy. However, for PE and PG, the intensity of the ion by loss of R₁CO₂H was stronger than that of ion by elimination of R₂CO₂H. These fragmentation rules could be used to determine the position of the fatty-acyl substituent of glycerol backbone.