New phenyl and thiophene dispiro indeno quinoxaline pyrrolidine quinolone analogues were synthesized by a one-pot four-component [3+2] cycloaddition reaction between (E)-3-arylidene-2,3-dihydro-8-nitro-4-quinolones,o-phenylenediamine, ninhydrin, and benzylamine/thiophenemethylamine. The structure and regio- and stereochemistry of the compounds have been confirmed by various spectroscopic and X-ray diffraction analyses. The observed regio- and stereoselectivity of the compounds were further enlightened by DFT calculations. Further, thein vitrocytotoxic effects of the compounds were evaluated by MTT assay. The compounds exhibited significant inhibition activity towards the tested cancer cells HeLa and MCF-7. Among the synthesized compounds compound9eshowed the highest inhibitory potency against MCF-7 cells. Mechanistic cell death studies revealed that cell death was induced by9eagainst MCF-7 cells by ROS mediated G1/S and G2/M phase cell cycle arrest apoptosis. The apoptotic cell death was quantified by the annexin V-FITC/PI staining method. The binding mode of compound9ewas further examined byin silicomolecular docking studies. © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2020.