The lipid backbones of sphingolipids and their metabolites are highly bioactive compounds that affect diverse cellular functions. The metabolites that have been most extensively studied with respect to their effects on cell behavior are ceramides, sphingosine (and other sphingoid bases), and sphingosine 1-phosphate. Additionally, there is interest in other naturally occurring species such as lysosphingolipids (sphingosine, phosphorylcholine, and psychosines) and N-methyl (di- and tri-methyl)-sphingosines. In many cases, studies of cell signaling mediated by these compounds have focused on a single category (such as ceramides or sphingosine 1-phosphate) because of the technical difficulty of more comprehensive analyses. One obstacle in such studies is that most of these compounds are metabolically interconvertable, so it is difficult to assign a conclusive relationship. In this article, we describe the analytical methods for extraction, identification, and quantitation of sphingolipids using state-of-the-art tandem mass spectrometry (MS/MS). Precursor ion scans are used to distinguish various species of sphingolipids in crude extracts by their unique molecular decomposition products. Specific headgroup, sphingoid base, and fatty acid chain combinations can be readily determined. Quantitation is achieved by multiple reaction monitoring (MRM) in conjunction with high-pressure liquid chromatography (HPLC). Compared to precursor ion scans alone, MRM experiments yield greater sensitivity and lower limits of detection by monitoring a specific precursor and product ion pair. This sensitivity facilitates detection of dozens of individual molecular species under optimal ion formation and decomposition conditions for each species, eliminating any ambiguity that may arise from differences in the kinetics of dissociation.