Unonopsis (Annonaceae) is a Neotropical genus with broad distribution through the Amazon Region. Within Unonopsis, some species, e.g. U. stipitata, have been described in traditional medicine to treat age related cognitive disorder. Previous studies indicated U. stipitata as a promising source of aporphine alkaloids, being this class extensively explored due to the biological activities previously reported [1-2]. Besides its attractive biological properties, the fast recognition and dereplication of this class in complex matrices by mass spectrometry (MS) have accelerated the research for new compounds. This favorable situation is possible due the advances on the gas-phase chemistry of isoquinoline-derived alkaloids (e.g. aporphine, oxoaporphine, tetradroprotoberberine and benzylisoquinoline skeletons) through key fragmentation recognition via collision induced dissociation (CID) experiments [3-4]. In order to screening potential acetylcholinesterase inhibitory alkaloids from U. stipitata, an integrative approach based on the leaf spray ionization multiple stage mass spectrometry (LS-MSn) and molecular docking was performed. A total of 8 isoquinoline-derived alkaloids (norjuziphine, reticuline, norglaucine, glaucine, oxoglaucine, N-formylnorglaucine, norisocorydine and nornantenine) were tentatively identificated, being the hypothetical binding modes between the most active compounds (norglaucine and oxoglaucine) and the acetylcholinesterase enzyme (AChE) suggested by molecular docking calculations. These data support previous pharmacological studies regarding isoquinoline-derived alkaloids with acetylcholinesterase inhibitory potential [5-6].