Lipidomics is a rapidly expanding research field that uses analytical chemistry to investigate 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 (i.e., shotgun lipidomics) 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 without the need for chromatographic purification. In this presentation, the principles of ESI intrasource separation that we have exploited for lipid analysis based on the inherent electrical propensities of different lipid classes will be briefly discussed. The application of shotgun lipidomics to biomarker discovery in Alzheimer’s disease (AD) (i.e., the specific depletion of sulfatide mass content in brain tissues and cerebrospinal fluid at the earliest clinically recognizable stage of AD) will be presented.