The lipidome is the precise complement of chemically distinct covalent lipid entities in a cell type or intact organ. Lipidomics refers to research investigating the biological function, significance, and sequelae of alterations in lipids and protein constituents mediating lipid metabolism, trafficking, or biological function in cells. Lipidomics has been greatly facilitated by recent advances in, and novel applications of, electrospray ionization mass spectrometry (ESI/MS). By employing ESI intrasource separation techniques that we have developed, individual molecular species of most major and many minor lipid classes can be fingerprinted and quantitated directly from biological lipid extracts. In this article, we briefly describe the principles of ESI intrasource separation that we have exploited for lipid analysis based on the inherent electrical propensities of different lipid classes. This strategy allows quantification of lipid molecular species directly from organic extracts of biological samples without the need for chromatographic purification. Examples of the application of this technology to lipid metabolism studies in two prevalent disease states (i.e., Alzheimer’s disease and diabetes) are given. Through appropriate utilization of ESI intrasource separation, the role of lipid alterations underlying disease states and the biochemical mechanisms through which lipids mediate cellular function in health and disease (i.e., functional lipidomics) can be further clarified. It is our hope that this knowledge will help us modify the deleterious consequences of lipidassociated diseases, such as diabetes, atherosclerosis, and obesity, afflicting industrialized countries in epidemic proportions.