Lipidomics is a rapidly expanding research field in which multiple techniques are utilized to quantitate the hundreds of chemically distinct lipids in cells and determine the molecular mechanisms through which they facilitate cellular function. Recent developments in electrospray ionization mass spectrometry (ESI/MS) have made possible, for the first time, the precise identification and quantification of alterations in a cell's lipidome after cellular perturbations. This review provides an overview of the essential role of ESI/MS in lipidomics, presents a broad strategy applicable for the generation of lipidomes directly from cellular extracts of biological samples by ESI/MS, and summarizes salient examples of strategies utilized to conquer the lipidome in physiologic signaling as well as pathophysiologically relevant disease states. Because of its unparalleled sensitivity, specificity, and efficiency, ESI/MS has provided a critical bridge to generate highly accurate data that fingerprint cellular lipidomes to facilitate insight into the functional role of subcellular membrane compartments and microdomains in mammalian cells. We believe that ESI/MS-facilitated lipidomics has now opened a critical door that will greatly increase our understanding of human disease.