Many enzymes and metabolites display difficult physicochemical properties that complicate their analysis in biological samples, and many metabolic pathways that enzymes regulate in a disease-specific context are not understood. These challenges can be addressed by applying innovative metabolomics and ABPP approaches to mapping biochemical pathways that support disease. Using selective inhibitors developed through competitive ABPP efforts or RNA interference technology, the function of an enzyme of interest can be specifically blocked, and then the metabolites that the enzyme regulates can be profiled. In this manner, not only can the substrates and products of an enzyme in specific (patho)physiological contexts be examined, but also the metabolic networks that the enzyme regulates can be identified and annotated. Collectively, this platform will allow identification of novel biochemical roles of already characterized enzymes, or may allow the identification of metabolic roles of completely uncharacterized enzymes.