A series of β-ionone derived chalcones were evaluated for cytotoxic activity against various human cancer cell lines using SRB dye assay. All the compounds displayed moderate to high cytotoxic effect against almost all the cancer cell lines. The results also revealed the effect of substituents of the aromatic ring on their inhibitory potential. In general, compounds bearing electron withdrawing groups such as nitro, fluoro, chloro and bromo showed more inhibitory potential than those bearing electron donating groups. The nitro substituted compound (7h) showed comparatively more inhibitory potential than other derivatives, therefore, it was further investigated for observing its effect on cell morphology in Chinese hamster ovary (CHO) cells by using phase contrast imaging, cell cycle analysis and annexin-FITC apoptosis assay. The treated cells exhibited the characteristics of apoptosis i.e. cell shrinkage, chromatin condensation and nuclear fragmentation, and induced the inhibition of cell proliferation by arresting the cells at G0 phase.