Dispiro andrographolide derivatives have been prepared from isatin/acenaphthoquinone, N-benzyl glycine and andrographolide via azomethine ylide cycloaddition reaction. The cytotoxic effect of the synthesized molecules has been studied against MCF-7 breast cancer cell line. The compounds induced apoptotic cell death as revealed by increased labeling with Annexin-V, decreased polarization of cell mitochondria, and increased reactive oxygen species production. Using FACS and western blot analysis, the compounds were observed to block the cell cycle at S phase. Activation of caspases 7 and 9 suggested that caspase pathways were involved in inducing apoptosis.