Engineering of specialized metabolites in plants, microbes, and other organisms is hindered by significant knowledge gaps about metabolic pathways responsible for metabolite accumulation and degradation. While isotopic tracers have provided important information about metabolic fluxes in central metabolism, limitations of mass spectrometric strategies for quantifying stable isotope incorporation into both intact metabolites and specific substructures have slowed extension of these techniques to large specialized metabolites. This report describes the application of electrospray ionization with data-independent multiplexed nonselective collision induced dissociation (CID) on a time-of-flight mass spectrometer. This strategy yields quasi-simultaneous collection, on the chromatographic time scale, of mass spectra with different degrees of fragment ion formation without biases introduced by precursor mass selection or selective ion activation and provides measurements of stable isotope enrichments in intact metabolites and individual substructures. The utility and precision of these analyses is demonstrated by labeling acylsugar metabolites in glandular trichomes of tomato (Solanum lycopersicum) using (13)CO2 and analyzing (13)C enrichments in acylsugar specialized metabolites. The high precision and avoidance of mass bias provide a promising tool for extending metabolic flux analyses to complex specialized metabolites in a wide range of organisms.