Glucosinolates, which are unique to Brassicaceae vegetables, have diverse biological activities, including antimicrobial, antioxidant, and anticancer actions. In this study, we applied hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-MS/MS) to the simultaneous quantification of 22 glucosinolates in 12 Brassicaceae vegetables, including pak choi, choy sum, Chinese cabbage, cauliflower, cabbage, broccoli, Kai Lan, Brussels sprouts, rocket salad, daikon radish, red cherry radish, and watercress. Significant differences in concentration and composition of glucosinolates were observed among these vegetables. Cabbage had the highest level of total glucosinolates (μg/g dry weight: 19 551.2 ± 1317.7), whereas Kai Lan had the lowest level (7611.3 ± 868.4). Aliphatic and indole glucosinolates were the major components in the 12 vegetables ranging from 76 to 100%, except watercress (37%). On the basis of the content of glucosinolates, the 12 vegetables were well distinguishable and classified according to their morphological taxonomy. This study presents a HILIC-MS/MS approach for quantification of glucosinolates, and demonstrates the potential of glucosinolate profiles for Brassicaceae species identification.