Metabolomic profiling studies aim to provide a comprehensive, quantitative, and dynamic portrait of the endogenous metabolites in a biological system. While contemporary technologies permit routine profiling of many metabolites, intrinsically labile metabolites are often improperly measured or omitted from studies due to unwanted chemical transformations that occur during sample preparation or mass spectrometric analysis. The primary glycolytic metabolite 1,3-bisphosphoglyceric acid (1,3-BPG) typifies this class of metabolites, and, despite its central position in metabolism, has largely eluded analysis in profiling studies. Here we take advantage of the reactive acylphosphate group in 1,3-BPG to chemically trap the metabolite with hydroxylamine during metabolite isolation, enabling quantitative analysis by targeted LC-MS/MS. This approach is compatible with complex cellular metabolome, permits specific detection of the reactive (1,3-) instead of nonreactive (2,3-) BPG isomer, and has enabled direct analysis of dynamic 1,3-BPG levels resulting from perturbations to glucose processing. These studies confirmed that standard metabolomic methods misrepresent cellular 1,3-BPG levels in response to altered glucose metabolism and underscore the potential for chemical trapping to be used for other classes of reactive metabolites.