Although lipids participate in many cellular processes both as signaling and structural molecules, our understanding of the roles of individual lipids as well as global changes in lipid composition are limited. Here we describe an LC-MS based method to identify lipids that change in a biological process. This method describes the isolation of lipids from tissue culture cells, sample preparation for LC-MS, the LC-MS run, and the subsequent data processing steps to compare the global lipid profiles and identify species that are enhanced or depleted. Identifying lipids that change is the first step towards functional studies to unravel their roles.