Tubulin is an attractive and established target for anticancer therapy. To date, the only method to determine the binding of inhibitor to tubulin has been competitive radioligand binding assays. We developed a non-radioactive mass spectrometry (MS) binding assay to study the tubulin binding of colchicine, vinblastine and paclitaxel and to identify which of these three binding sites that a novel inhibitor binds. The method involves a very simple step of separating the unbound ligand from macromolecules using ultrafiltration. The unbound ligand in the filtrate can be accurately determined using highly sensitive and specific liquid chromatography tandem mass spectrometry (LC-MS/MS) method using multiple reaction monitoring (MRM) mode. The assay was validated using podophyllotoxin, vincristine and docetaxel, drugs that compete to the colchicine-, vinblastine- and paclitaxel-binding sites in tubulin, respectively. This competitive binding assay allowed the reliable detection of interactions of these drugs with three binding sites on tubulin. This method was subsequently applied to determine the tubulin-binding site of 4-substituted methoxylbenzoyl-aryl-thiazoles (SMART-H), a potent antitubulin agent developed in our laboratory. The results indicated that SMART-H specifically and reversibly bound only to the colchicine-binding site, but not to vinblastine- or paclitaxel sites. This new non-radioligand binding method to determine the binding site on tubulin will function as a useful tool to study the binding sites of tubulin inhibitors. Copyright © 2010 John Wiley & Sons, Ltd.