Recently, Zheng et al. identified and characterized a novel tubulin polymerization inhibitor discovered during anti-cancer compound screenings using the epithelial-mesenchymal transition (EMT)-mimetic assay. The lead compound was a nitrobenzoate molecule (2-morpholin-4-yl-5-nitro-benzoic acid 4-methylsulfanyl-benzyl ester), designated as compound IMB5046. As part of the in vitro and in vivo characterization process, in vitro tubulin polymerization assays with 97% tubulin/3% microtubule-associated proteins (MAPs) under cell-free conditions demonstrated that the compound inhibits tubulin polymerization, complementing findings from cell culture and mouse xenograft model studies. In addition, surface plasmon resonance (SPR) technology quantified the binding interaction of IMB5046 and tubulin, using biotinylated tubulin and streptavidin-coated sensor chips. SPR data demonstrated a concentration-dependent, direct interaction and an equilibrium dissociation constant (Kd) of 31.9 µM for IMB5046. Cytoskeleton’s HTS-tubulin polymerization assay kit and >99% pure biotinylated porcine brain tubulin (Cat.# BK004P and T333P, respectively) were essential in this study, providing the necessary research tools to measure the effect of IMB5046 on in vitro tubulin polymerization as well as the binding kinetics with tubulin. In combination with the other findings, a unique, anti-cancer compound with novel chemical structure and the ability to inhibit tubulin polymerization in multidrug-resistant cancer cell lines has been discovered and described.