This review describes the use of low-energy collisionally activated dissociation (CAD) with both tandem quadrupole and ion-trap mass spectrometry toward structural characterization of glycerophospholipids (GPLs), including classes of glycerophosphocholine, glycerophosphoethanolamine, glycerophosphoserine, glycerophosphoglycerol glycerophosphoinositol and glycerophosphatidic acid, as well as their lyso-, plasmanyl-, and plasmenylphospholipid subclasses. The mechanisms underlying the fragmentation processes leading to structural characterization of GPLs in various ion forms desorbed by electrospray ionization in the positive-ion and negative-ion modes are also discussed. The tandem mass spectrometric approaches afford the identification of the polar head group, the fatty acid substituents and the location of the radyl groups on the glycerol backbone of all the GPLs.