Sphingolipids are an important class of structural and signaling molecules within the cell. As sphingolipids have been implicated in the development and pathogenesis of insulin resistance and the metabolic syndrome, it is important to understand their regulation and metabolism. Although these lipids are initially produced through a common pathway, there is no “generic” sphingolipid. Indeed, the biophysical and signaling properties of lipids may be manipulated by the subunit composition or isoform of their synthetic enzymes, via regulation of substrate integration. Functionally distinct pools of chemically-equivalent lipids may also be generated by de novo synthesis and recycling of existing complex sphingolipids. The highly integrated metabolism of the many bioactive sphingolipids means that manipulation of one enzyme or metabolite can result in a ripple effect, causing unforeseen changes in metabolite levels, enzyme activities, and cellular programmes. Fortunately, a suite of techniques, ranging from thin-layer chromatography to liquid chromatography-mass spectrometry approaches, allows investigators to undertake a functional characterization of all or part of the sphingolipidome in their systems of interest.