Although the genus Duguetia is well known for producing alkaloids as chemical constituents, there are no reports of alkaloids identified in the species D. lanceolata. Thus, aiming to identify the chemical composition of this species, the dereplication of alkaloidic phase was performed by use of ultra-high performance liquid chromatography high resolution electrospray ionization tandem mass spectrometry (UHPLC-HR-ESI-MS/MS) and H-1 nuclear magnetic resonance (NMR). The chromatographic fractionation of the alkaloid extract from Duguetia lanceolata (Annonaceae) leaves afforded four fractions (I-IV) that were shown to be composed of aporphine alkaloids. H-1 NMR analysis and UHPLC-HR-ESI-MS/MS based dereplication allowed the identification of eight alkaloids: glaucine (1). norglaucine (2), isocorydine (3). N-methyllaurotetanine (4), oxoglaucine (5), liriodenine (6), lanuginosine (7), dehydroglaucine (8). Compounds 2, 3, 4, 6 and 7 were described for the first time in this species, while alkaloids 1, 5 and 8 are newly discovered in the genus Duguetia. Additionally. the antiparasitic activity of the four fractions was evaluated in vitro against Leishmania infantum and Trypanosoma cruzi. Fraction I, composed exclusively by 1, displayed activity against Leishmania infant= and Trypanosoma cruzi intracellular amastigotes, with half maximal inhibitory concentration (IC50) values of 7.5 and 28.6 mu g mL(-1), respectively. Fraction IV (constituted by 2, 3 and 4) showed activity against promastigotes of Leishmania infantum with IC50 value of 50.0 mu g mL(-1),while fraction II (constituted by 5 and 6) showed activity against trypomastigotes of Trypanosoma cruzi, with IC50 values of 83.0 mu g mL(-1). This work showed that fragmentation in UHPLC-HR-ESI-MS/MS combined with H-1 NMR analysis of fractions is useful for identifying alkaloids in mixtures. Additionally, it was also demonstrated the potential of aporphine alkaloids from Duguetia lanceolata St.-Hil (Annonaceae) in the search for new drug candidates for neglected diseases.