Flavonoids are one of the largest groups of secondary metabolites and are present in plants as aglycones linked to glycosides. LC-MSMS experiments are used in this work to establish the flavonoid content of Euphorbia hirta L. Based on the identification of aglycone ions produced upon collision-induced dissociation (CID), the structural characterization of each flavonoid ion is finally achieved based on the MSMS data. Fourteen flavonoids presenting five different aglycones: quercetin, myricetin, kaempferol, luteolin, and apigenin, are identified in the ethyl acetate extract of the plant. Among all the observed flavonoids, myricetin-3-O-glucoside, myricetin-7-O-glucoside, kaempferol-3-O-glucoside, vitexin, and isovitexin are detected for the first time in the plant extract. The distinction between vitexin and isovitexin is achieved using ion mobility mass spectrometry. Quantitative evaluation shows that Euphorbia hirta extract contains, 136 ± 2, 64 ± 2 and 6.9 ± 0.3 μg of quercitrin, myricitrin and quercetin per mg of ethyl acetate extract, respectively.