The discovery and optimization of a series of 2-N-aryl-substituted benzenesulfonamidoacetamides as novel tubulin polymerization inhibitors are described. Pharmacophore exploration of hit compound AH-487 identified the optimal structure of N-heteroaryl-2-(4-methoxy-N-(3-(trifluoromethyl)phenyl)phenylsulfonamido)acetamide as a potent antimitotic agent. Subsequent lead compounds 4 b and 4 c, with N-4-aminophenyl and N-1H-indol-5-yl substitutions at the acetamide position, respectively, were shown to induce cell-cycle arrest at the G2/M phase and lead to an accumulation of HeLa cells in the sub-G1 phase. More significantly, these lead compounds (3 c, 4 b, and 4 c) exhibit impressive cytotoxicity against a panel of cancer cells including P-glycoprotein-overexpressing MDR-positive cells, with potency greater than or equal to clinically studied benzenesulfonamide E7010. Mechanistic studies demonstrated that derivatives of AH-487 disrupt mitotic spindles by inhibiting microtubule polymerization and induce apoptosis via induction of Bcl-2 phosphorylation in tumor cells. The optimized leads 4 b and 4 c strongly inhibited the growth of human hepatocellular carcinoma cells in a mouse xenograft model.