Based on classical colchicine site ligands and a computational model of the colchicine binding site on beta tubulin, two classes of chalcone derivatives were designed, synthesized and evaluated for inhibition of tubulin assembly and toxicity in human cancer cell lines. Docking studies suggested that the chalcone scaffold could fit the colchicine site on tubulin in an orientation similar to that of the natural product. In particular, a 3,4,5-trimethoxyphenyl ring adjacent to the carbonyl group appeared to benefit the ligand-tubulin interaction, occupying the same subcavity as the corresponding moiety in colchicine. Consistent with modeling predictions, several 3,4,5-trimethoxychalcones showed improved cytotoxicity to murine acute lymphoblastic leukemia cells compared with a previously described parent compound, and inhibited tubulin assembly in vitro as potently as colchicine. The most potent chalcones inhibited the growth of human leukemia cell lines at nanomolar concentrations, caused microtubule destabilization and mitotic arrest in human cervical cancer cells, and inhibited human breast cancer cell migration in scratch wound and Boyden chamber assays.