Unexpectedly, we found that the C-MST assay could detect tubulin interactions with ligands targeting other tubulin-binding sites. This capability was demonstrated using a diverse set of reference ligands, including vinblastine, pironetin, ansamitocin P3, maytansine, or a natural tubulin ligand, guanosine diphosphate (GDP) (Fig. ). Interestingly, all alternative-site binders displayed signals below the control level, contrasting with colchicine-site binders. This distinction enables a clear differentiation of binding sites based on the screening output (Fig. , Fig. ). We attribute these differences to the distinct nature of the signals: colchicine-site binders displace coumarin-30 from tubulin, while ligands targeting alternative binding pockets alter the thermophoresis of the tubulin-coumarin-30 complex (see Supporting Information for further analysis of this phenomenon).