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In this work an LC-MS/MS method has been developed for the simultaneous analysis of kasugamycin (KAS) and streptomycin (STR) in vegetable samples. The use of a mixed-mode HPLC column and a ternary mobile phase acetonitrile:water:0.5% aqueous formic acid in gradient elution mode are proposed to provide enough retention and resolution for these highly polar compounds. Heated-electrospray ionization (H-ESI) has been used to ionize KAS and STR when coupling liquid chromatography to mass spectrometry. In contrast to what happens with most aminoglycosides, KAS only showed singly charged ions in the full-scan mass spectrum. The [M + H]+ of both KAS and STR are proposed as precursor ions for tandem mass spectrometry since more stable product ions and better ion ratio precisions are obtained. Fragmentation of KAS has been studied, showing that the cleavage of the glycosidic bonds provided the main product ions and the most abundant and selective ones are proposed for quantitation and confirmation purposes in MRM mode. The LC-MS/MS method developed has been applied to the analysis of vegetables. A simple clean-up procedure using hydrophilic–lipophilic-balanced cartridges was applied to several vegetable extracts from tomato, zucchini, chard and lettuce, obtaining recoveries >80% for both compounds. The high matrix suppression observed for KAS in all matrices was reduced by a 1 : 5 sample dilution with acetonitrile, providing a 100-fold improvement in sensitivity for this compound. Method quality parameters have been established for tomato matrices, obtaining method limits of quantitation in the low µg kg−1 level (5–10 µg kg−1) for both compounds, precisions expressed in %RSD better than 7% and accuracies expressed as relative error better than 8% at the 100 µg kg−1 level, making this method suitable for routine analysis.