Ageratum conyzoides L. is a plant widely used in traditional medicine of tropical and subtropical regions for its anti-inflammatory and antinociceptive properties. Nevertheless, the chemical composition of its medicinal preparation has not been yet accurately established. In this study, chromatographic methods were developed for the simultaneous identification and quantification of the main compounds in A. conyzoides aqueous extract, using UPLC-PDA-ESI-QToF-MS. The qualitative analyses defined by MS/MS analysis allowed the identification of 27 compounds in the aqueous extract, including the toxic pyrrolizidine alkaloids, phenolic acids, coumarin and polymethoxyflavones. Among the metabolites, twelve were detected for the first time in the species. The quantitative method was validated according to the official guidelines, demonstrating to be specific, linear, precise, accurate and sensitive for the quantification of chlorogenic acid, coumaric acid, coumarin (1,2-benzopyranone), 5,6,7,3′,4′,5′-hexamethoxyflavone, nobiletin, 5′-methoxynobiletin and eupalestin.