Cells signal through lipids that are produced by phospholipid (PL) and phosphoinositide (PIPn) metabolism involve three enzymatic processes: (i) ester and phosphodiester hydrolysis by phospholipases, (ii) monophosphate hydrolysis by phosphatases, and (iii) phosphorylation of hydroxy groups by kinases. Unregulated enzyme activity correlates with specific pathologies, which are specific targets for therapeutic intervention. A variety of reagents now permit monitoring of in vitro enzyme activity, spatiotemporal changes of intracellular lipid concentrations, and identification of lipid-protein interactions. This minireview summarizes a chemical biology approach that illustrates how chemically synthesized affinity probes can be used to characterize changes in lipid signaling in cellular and molecular biology.