A phenotypic high-throughput screen identified a benzamide small molecule with activity against small cell lung cancer cells. A "clickable" benzamide probe was designed that irreversibly bound a single 50 kDa cellular protein, identified by mass spectrometry as β-tubulin. Moreover, the anti-cancer potency of a series of benzamide analogs strongly correlated with probe competition, indicating that β-tubulin was the functional target. Additional evidence suggested that benzamides covalently modified Cys239 within the colchicine binding site. Consistent with this mechanism, benzamides impaired growth of microtubules formed with β-tubulin harboring Cys239, but not β tubulin encoding Ser239. We therefore designed an aldehyde-containing analog capable of trapping Ser239 in β tubulin, presumably as a hemiacetal. Using a forward genetics strategy, we identified benzamide-resistant cell lines harboring a Thr238Ala mutation in β-tubulin sufficient to induce compound resistance. The disclosed chemical probes are useful to identify other colchicine site binders, a frequent target of structurally diverse small molecules.