Biosynthesis and release of small lipid molecules from cells is a common strategy that has evolved in multicellular organisms in order to signal nearby cells and coordinate biochemical events within cells in the immediate microenvironment. This biochemical coordination with the target cell is achieved by specific signalling proteins such as cell surface G-protein linked receptors that recognize these small lipid molecules. Many of these signal-inducing lipids are metabolites of arachidonic acid and have come to be called by the generic name “eicosanoids.” Tandem mass spectrometry has played a central role not only in the initial structural identification of these lipids, but also in the quantitation of very low levels of all eicosanoids, eicosanoid metabolites, and other lipid mediators. This chapter will deal only with the ion chemistry of eicosanoids, their metabolites, and other lipid mediators.