Background: Vepris dainelli (Rutaceae) is an endemic medicinal plant of Ethiopia, traditionally used for the treatment of abdominal cramps, intestinal worms, skin diseases, and tooth pain. Methods: Roots and fruit extracts were subjected to silica gel column chromatographic separation to afford five alkaloids, reported for the first time from the species. The cytotoxic effects of alkaloids (2-4) were evaluated in vitro against the estrogen-responsive MCF-7 and estrogen-unrespon-sive MDA-MB-231 human breast cancer cell lines by MTS assay. Results: The results revealed that alkaloids (2-4) induced a significant reduction in cell growth of both breast cancer cell lines in a dose-dependent manner. Evodiamine (4) showed the highest poten-cy against the aggressive metastatic MDA-MB-231 cell line at low micromolar concentrations. In addition, it highly arrested the cells in the G2/M phase, especially the MCF-7 cell line. By contrast, evoxanthine (2) and arborinine (3) exhibited higher cytotoxicity against MCF-7 than MDA-M-B-231 and influenced the cell cycle in both cell lines by arresting some cells in the G2/M phase, preventing cells with damaged DNA from entering mitosis. Molecular docking analysis showed that all alkaloids inhibit human topoisomerase II α, compared with vosaroxin’s anti-cancer agent under clinical trial. The ADMET studies revealed that the alkaloids showed the highest drug-like-ness properties, suggesting that these alkaloids act as a drug and exhibit remarkable biological ac-tivities, except (5). DFT calculations indicated that the studied alkaloids showed the lowest gap en-ergy and were chemically reactive. Conclusion: The results obtained from molecular docking, drug-likeness properties, ADMET anal-ysis, and DFT calculation are in good agreement with experimental studies. Hence, evoxanthine (2), arborinine (3), and evodiamine (4) may serve as lead molecules that could be developed into potent topoisomerase II α inhibitors against human breast cancer cells. © 2022 Bentham Science Publishers.