A new series of 2-arylaminobenzothiazole-arylpropenone conjugates 5–6(a–r) was designed, synthesized and investigated for their cytotoxic potency against the various human cancer cell lines. Most of these conjugates exhibited cytotoxic activity and inhibited in vitro tubulin polymerization effectively. Conjugates 5d and 6d cause cell cycle blocks in the G2/M phase in HeLa cells and treatments with 5d and 6d manifested increased mRNA and protein levels of the G2/M marker, cyclin B1. Immunocytochemistry revealed loss of intact microtubule structure in cells treated with 5d and 6d. Western blot analysis revealed that these conjugates accumulate more tubulin in the soluble fraction. Moreover, the triggering of apoptotic cell death after mitotic arrest was investigated by studying their effect on Hoechst staining, mitochondrial membrane potential, ROS generation.