A series of new 4β-sulphonamido and 4β-[(4'-sulphonamido)benzamide] conjugates of podophyllotoxin (11a-j and 15a-g) were synthesized and evaluated for anticancer activity against six human cancer cell lines and found to be more potent than etoposide. Some of the compounds 11b, 11d and 11e that showed significant antiproliferative activity in Colo-205 cells, were superior to etoposide. The flow cytometric analysis indicates that these compounds (11b, 11d and 11e) showed G2/M cell cycle arrest and among them 11e is the most effective. It is observed that this compound (11e) caused both single-strand DNA breaks as observed by comet assay as well as double-strand DNA breaks as indicated by γ-H2AX. Further 11e showed inhibition of topo-IIα as observed from Western blot analysis and related studies. Compounds caused activation of ATM as well as Chk1 protein indicating that the compound caused effective DNA damage. Moreover activation of caspase-3, p21, p16, NF-kB and down regulation of Bcl-2 protein suggests that this compound (11e) has apoptotic cell death inducing ability, apart from acting as a topo-IIα inhibitor.