A new series of tetrazole based isoxazolines (4a–l) was synthesized and evaluated for their anticancer potential against two cancer cell lines. All these compounds exhibited profound cytotoxicity with IC50 values ranging from 1.22 to 3.62 mM and compounds 4h, 4i showed prominent anticancer efficacy with IC50 values of 1.51, 1.49 mM in A549 and 2.83, 2.40 mM in MDA-MB-231 cell lines. Further, these compounds (4h, 4i) induced apoptotic cell death by inhibition of tubulin polymerization leading to cell cycle arrest at G2/M phase of the cell cycle followed by caspase-3 activity. Moreover, the level of tubulin inhibition by these compounds was examined by in vitro HTS tubulin polymerization assay. Docking of compound 4h and 4i to the active site of tubulin revealed that the trimethoxy ring of the compounds occupies the colchicine binding site of tubulin, whereas the isoxazoline moiety moves towards the interface of a–b tubulin and involves a series of hydrogen bonds with aTyr224 and aSer178.