Benzoxazinoids (BXs) are phytochemicals that exert plant-protecting, allelopathic, and human-health-promoting effects. Here, we present a data-dependent acquisition-mass spectrometry based method to locate and annotate BX-conjugates in different fractions of a plant extract. This allows for isolation of compounds present in a much smaller percentage than common for natural products yet still sufficient for subsequent identification through nuclear magnetic resonance (NMR) spectroscopy. The presence of BXs has been extensively studied in monocotyledons, whereas only a limited number of studies have focused on dicotyledons. Here, the presence of hitherto unknown BXs in the roots of the dicotyledonous plant Acanthus mollis L. has been determined. Two acetylated glycosylated BXs, compound 1 2-(6-acetyl-β-D-glucopyranoside)-2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one) and compound 2 (-2-(6-acetyl-β-D-glucopyranoside)-2-hydroxy-2H-1,4-benzoxazin-3(4H)-one), and a BX-derived glucoside carbamate olide, compound 3 (2-hydroxy-6-(2-deoxy-β-D-glucopyranosyloxy)-phenyl) carbamic acid 2′-olide), were isolated and identified by NMR. These previously unknown BXs may contribute to the reported biological activities of A. mollis L.