In this paper, UHPLC-DAD-MS/MS analysis was used for determination of the glucosinolate profile of various plant parts of Lepidium draba and an unknown plant sample, as well as the glucosinolate profile of Sinapis alba seeds. Glucosinolates were isolated from the samples in their desulfated form using a modified ISO 9167-1 method. The obtained glucosinolate profile of Lepidium draba was compared with the glucosinolate profile of the unknown plant species in order to determine chemotaxonomic characteristics of the unknown plant sample. In both samples, the highest glucosinolate content was found in the leaves, while the lowest content was found in the stem. The most abundant compound in the leaves of both samples was glucoraphanin (41.49 ± 4.05 μmol/g dry plant material of Lepidium draba and 40.40 ± 3.62 μmol/g dry plant material of the unknown sample) and glucosinalbin in the inflorescence (32.03 ± 5.43 μmol/g in Lepidium draba and 34.73 ± 0.76 μmol/g in the unknown sample). The most abundant glucosinolates in both samples are derived from methionine. Based on the significant similarity in the glucosinolate profiles and UV and MS2 spectra of the samples, it was concluded that the unknown sample likely belongs to the Lepidium draba species. Since the glucosinolate profile of Sinapis alba seeds was much simpler than that of the above-mentioned samples, this sample was used for confirmation of the structure of the most abundant compound in the seeds - desulfoglucosinalbin. The structure was confirmed using NMR techniques, and the β configuration of the anomeric carbon atom of the glucose unit of glucosinalbin was determined.