Background: Extensive bioactivities of alkaloids from the genus Macleaya (Macleaya cordata (Willd.) R. Br. and Macleaya microcarpa (Maxim.) Fedde) have been widely reported, as well as more and more concerned from the scientific communities. However, systematic research on the phytochemical information of M. microcarpa is incomplete. The aim of this study was to rapidly and conveniently qualitative analyze alkaloids from M. microcarpa by ultra-perfor-mance liquid chromatography/quadrupole-time-of-fight mass spectrometry (UHPLC–Q-TOF-MS) using accurate mass weight and characteristic fragment ions, furthermore separate and identify the main alkaloids, test antitumor activity in vitro and antiangiogenic activity in vivo. Results: A total of 14 alkaloids from fruits of M. microcarpa were identified by UHPLC–Q-TOF-MS, including 5 pro-topines, 2 benzophenanthridines, 1 dimer, 1 dihydrobenzophenanthridines and 5 unknown structure compounds. Two major alkaloids were isolated by various column chromatographic methods. Their structures were determined by NMR data and related literatures. The two major alkaloids were evaluated for intro cytotoxic activities against HL-60, MCF-7, A-549, and in vivo antiangiogenic activity using transgenic zebrafish. Conclusions: Current qualitative method based on UHPLC–Q-TOF-MS technique provided a scientific basis for isolation, structural identification, and in vitro or in vivo pharmacological further study of alkaloids from M. microcarpa in the future. © The Author(s) 2020.