A fundamental goal of proteomics is to assign physiological functions to all proteins encoded by eukaryotic and prokaryotic genomes. Of the many activities performed by proteins, the chemical transformations catalyzed by enzymes form the basis for most, if not all, metabolic and signaling pathways. Elucidation of these pathways and their integration into larger cellular networks require new strategies to rapidly and systematically identify physiological substrates of enzymes. Here, we review emerging technologies that aim to assign endogenous biochemical functions to enzymes by profiling the metabolome.