Using the C-MST assay and the coumarin-30 fluorescence enhancement assay as a complementary technique for validating the results, we identified several novel tubulin-binding compounds from the NCI library: cc-3, cc-4, gc-1, gc-2. We also developed an efficient synthetic procedure for benzo[d]pyrrolo[1,2-a]imidazoles 4, structural analogs of cc-4, and evaluated them in screening, finding a hit compound 4c. All of these substances were classified by the C-MST assay as colchicine site binders, including the spiro-compounds gc-1 and gc-2, which are structurally distinct from any known colchicine site binders. The inhibitory activities and cellular potencies of these compounds were further confirmed by optical microscopy and flow cytometry in vitro and in cancer cell culture, highlighting the utility of the coumarin-30-based assay in drug discovery.