Reactive electrophilic intermediates such as coenzyme A esters play central roles in metabolism but are difficult to detect with conventional strategies. Here, we introduce hydroxylamine-based stable isotope labeling to convert reactive electrophilic intermediates into stable derivatives that are easily detectable <i>via</i> LC-MS. In the model system <i>Caenorhabditis elegans</i>, parallel treatment with <sup>14</sup>NH<sub>2</sub>OH and <sup>15</sup>NH<sub>2</sub>OH revealed >1000 labeled metabolites, e.g., derived from peptide, fatty acid, and ascaroside pheromone biosyntheses. Results from NH<sub>2</sub>OH treatment of a pheromone biosynthesis mutant, <i>
acox-1.1</i>, suggested upregulation of thioesterase activity, which was confirmed by gene expression analysis. The upregulated thioesterase contributes to the biosynthesis of a specific subset of ascarosides, determining the balance of dispersal and attractive signals. These results demonstrate the utility of NH<sub>2</sub>OH labeling for investigating complex biosynthetic networks. Initial results with <i>Aspergillus</i> and human cell lines indicate applicability toward uncovering reactive metabolomes in diverse living systems.