Due to their broad-spectrum bioactivities and compliance with directed functionalization, pentacyclic triterpenoids (PCTs) are considered a potent molecular platform for novel drug development. Cytotoxic and pro-apoptotic activities of PCTs involving different specific mechanisms are of substantial interest in preferable targeting cancer cells. Replacement of С-3 hydroxyl group of ursolic acid by the nitrogen-containing group was earlier found to promote cytotoxicity of the triterpenoid. In this work, we studied and compared cytotoxicity of pre-synthesized and characterized lupane triterpenoid derivatives containing hydroxyl, amino or carboxymethyl amine groups at C-3 position. According to resazurin assay upon 3-day culture, the modified PCT showed increased inhibitory effect on viability of breast and prostate cancer cells (MCF-7 and PC-3 lines) with half-maximum inhibitory concentrations in the micromolar range. According to flow cytometry analysis with reactive oxygen species (ROS)-specific fluorescent probes, the synthesized compounds exhibited modulating concentration-dependent effects on cytoplasm and mitochondrial ROS levels; the most active derivative caused significant ROS overproduction, which probably underlies its cytotoxic effect. The obtained results contribute to the design of PCT derivatives with potential anticancer properties and encourage further investigation of the compounds. The study was supported by the Subsidy Allocated to Kazan Federal University for the State Assignment in the Sphere of Scientific Activities (project FZSM-2025-0002).