Four novel α-cyano-indolylchalcone derivatives ( 3a, 3b, 5a, and 5b) were synthesized, and their chemical structures were comprehensively characterized using various spectroscopic analytical techniques. The cytotoxicity of the synthesized compounds was evaluated using the MTT assay against different cancer cell lines, including breast cancer (MCF7), hepatocellular carcinoma (HepG2), and colon cancer (HCT116). Among the tested derivatives, compounds 5a and 5b exhibited the highest cytotoxic activity, demonstrating significant potency against HepG2 cells with IC50 values of 49.01 and 21.89 µg/mL, respectively. Furthermore, various molecular techniques were employed to investigate the apoptotic efficacy of compounds 5a and 5b in hepatocellular carcinoma cells.