The combination of high-resolution LC-MS-based untargeted metabolomics with stable isotope tracing provides a global overview of the cellular fate of precursor metabolites. This methodology enables detection of putative metabolites from biological samples and simultaneous quantification of the pattern and extent of isotope labeling. Labeling of Trypanosoma brucei cell cultures with 50% uniformly 13C-labeled glucose demonstrated incorporation of glucose-derived carbon into 187 of 588 putatively identified metabolites in diverse pathways including carbohydrate, nucleotide, lipid, and amino acid metabolism. Labeling patterns confirmed the metabolic pathways responsible for the biosynthesis of many detected metabolites, and labeling was detected in unexpected metabolites, including two higher sugar phosphates annotated as octulose phosphate and nonulose phosphate. This untargeted approach to stable isotope tracing facilitates the biochemical analysis of known pathways and yields rapid identification of previously unexplored areas of metabolism. © 2012 American Chemical Society.