The development of a top-down shotgun-MS method utilizing gas phase ion/ion charge inversion chemistry provides near-complete glycerophospholipid (GPL) structural identification as described previously. To date, few approaches are capable of discerning methyl-branching site in complex lipid structures. We report an MS n experiment involving ion/ion chemistry to facilitate the assignment of the GPL headgroup and fatty acyl composition, identification of the presence of anteiso methyl branching position in saturated acyl chains, and, in some cases, assignment of fatty acyl sn-position. To demonstrate this approach, we examined synthetic phosphatidylcholines (PCs), including PC 16:0/14:0(12Me) and PC and suggests preferential formation of the carboxylate anion from the sn-2 acyl substituent. Importantly for unknown lipids, fatty acyl chain regiochemical assignments based only on relative abundances of the carboxylate anions alone are insufficient without calibrating to standards and should be made with caution. However, it can provide a useful guide to the dominant regiochemistry i.e. the most abundant regioisomer.