In this study glucosinolates from the plant species Hesperis laciniata, Thlaspi perfoliatum (Brassicaceae), and Moringa oleifera (Moringaceae) were analyzed. The main goal was the isolation and identification of desulfoglucosinolates from different plant parts using a modified ISO 9167-1 method, followed by their qualitative and quantitative analysis using the UHPLC-DAD-MS/MS system. A total of seven different desulfoglucosinolates were identified, including several previously unknown compounds. The highest concentration of glucosinolates in H. laciniata was found in the flowers (110.08 µmol/g dry plant material), while in T. perfoliatum, the highest concentration was observed in the siliques (97.6 µmol/g). In the seeds of M. oleifera, the dominant compound was glucomoringin (221.74 µmol/g). In H. laciniata, the most abundant glucosinolate was 4´-O-β-D-apiofuranosylglucomatronalin (69.57 µmol/g dry plant material), whereas in T. perfoliatum, the dominant compound was 4´-O-(β-D- apiofuranosyloxy)benzyl-glucosinolate (53.86 µmol/g). Furthermore, successful isolation of individual glucosinolates was achieved using the HPLC-DAD system with a fraction collector. From T. perfoliatum, three fractions were isolated: desulfoglucosinalbin, desulfo-apiofuranosylglucomatronalin, and desulfo- (apiofuranosyloxy)benzyl-glucosinolate. The results highlight specific differences in glucosinolate profiles among the species studied, and the identified compounds represent a significant contribution to understanding the chemical diversity within the Brassicaceae and Moringaceae families. Notably, the identification of multiply glycosylated glucosinolates advances the characterization of the biological and chemical properties of these plants.