Purpose <p>This study aims to isolate and identify a biologically active lupane-type triterpenoid from the stem bark of <i>Vitex</i> <i>pubescens</i>, a traditional Indonesian medicinal plant. This research also investigates its potential cytotoxic activity against cancer cells, beginning with in silico analysis to evaluate interactions with cancer-related proteins. The findings from the in silico analysis are further validated through in vitro experiments using human cervical cancer cells (HeLa).</p> Methods <p>The stem bark of <i>V.</i> <i>pubescens</i> was extracted with methanol, yielding a crude extract that was fractionated with hexane, dichloromethane, and ethyl acetate. The dichloromethane fraction was further purified, isolating a lupane-type triterpenoid, identified via thin-layer chromatography with Liebermann–Burchard reagent and confirmed through UV, IR, and NMR analyses. In silico molecular docking was performed to evaluate the interactions of the lupane-type triterpenoid with cancer-related proteins (Bcl-2, Bcl-xl, Mcl-1, CDK-2, and TNIK). The potential cytotoxic activity of the compound was further confirmed by in vitro on HeLa cells using the MTT assay.</p> Results <p>The methanol extraction yielded 5.56% crude extract. Among fractions, ethyl acetate was predominant (42.80%). The isolated compound, identified as epibetulinic acid, demonstrated strong binding affinities with cancer-related proteins, with binding energies between −&#xa0;5.49 and −&#xa0;6.69&#xa0;kcal/mol. Epibetulinic acid showed significant inhibition against HeLa cells in a dose-dependent manner at 25, 50, and 100&#xa0;µg/mL concentrations, resulting in an IC₅₀ value of 51.98 ± 0.64&#xa0;µg/mL.</p> Conclusion <p>Epibetulinic acid from <i>V.</i> <i>pubescens</i> exhibits stable interactions with cancer-associated proteins and promising cytotoxic activity against cervical cancer, supporting its potential for further development in cervical cancer therapy.</p>