An analytical approach based on reversed-phase liquid chromatography coupled to electrospray ionization Fouriertransform mass spectrometry in negative ion mode (RPLC-ESI-(−)-FTMS) was developed for the untargeted characterization of glucosinolates (GSL) in the polar extracts of four Brassica microgreen crops, namely, garden cress, rapeseed, kale, and broccoli raab. Specifically, the all ion f ragmentation (AIF) operation mode enabled by a quadrupole-Orbitrap mass spectrometer, i.e., the systematic fragmentation of all ions generated in the electrospray source, followed by the acquisition of an FTMS spectrum, was exploited. First, the best qualifying product ions for GSL were recognized from higher-energy collisional dissociation (HCD)-FTMS 2 spectra of representative standard GSL. Extracted ion chromatograms (EIC) were subsequently obtained for those ions from RPLC-ESI(−)-AIF-FTMS data referred to microgreen extracts, by plotting the intensity of their signals as a function of retention time. The alignment of peaks detected in the EIC traces was finally exploited for the recognition of peaks potentially related to GSL, with the EIC obtained for the sulfate radical anion [SO 4 ] •− (exact m/z 95.9523) providing the highest selectivity. Each putative GSL was subsequently characterized by HCD-FTMS 2 analyses and by collisionally induced dissociation (CID) multistage MS n (n = 2, 3) acquisitions based on a linear ion trap mass spectrometer. As a result, up to 27 different GSLs were identified in the four Brassica microgreens. The general method described in this work appears as a promising approach for the study of GSL, known and novel, in plant extracts.