We validated the C-MST assay on a wide panel of well-established tubulin ligands of both the colchicine site and alternative sites. The method's high accuracy and sensitivity were further confirmed using a set of recently characterized synthetic 1,5-disubstituted pyrrolidin-2-ones and related derivatives. Finally, we used C-MST assay to characterize predicted antimitotics from the National Cancer Institute (NCI) library, identifying several potent binders of the colchicine site of tubulin. The results obtained were verified based on microtubule dynamics and cell cycle assays on cultured cancer cells.