In this work, we sought to combine the strengths of the MST approach, as a general-purpose method, with the advantages of using a target-specific fluorescent probe, in order to create an efficient, sensitive and informative assay for characterizing the binding of small molecules to tubulins. α/β Tubulins are essential heterodimeric proteins that form multi-tasking cytoskeletal structures known as microtubules. Microtubules exhibit remarkable dynamic behavior, characterized by intermittent assembly and disassembly, with stochastic transitions between these phases, termed catastrophes and rescues. This "dynamic instability" of microtubules is tightly regulated throughout the cell cycle to fine-tune the behavior of these polymers for their numerous cellular functions, including maintaining cell shape, positioning organelles, interacting with other cytoskeletal filaments, and facilitating chromosome capture by the mitotic spindle, and then chromosome segregation during cell division. Cytotoxic compounds that inhibit microtubule dynamics interfere with the proliferation of rapidly dividing cancer cells, leading to mitotic arrest often followed by apoptosis. Targeting tubulins with small molecule drugs remains a crucial strategy in modern cancer chemotherapy. Numerous drugs that bind various pockets on tubulins have been identified; over 20 tubulin-binding drugs are currently being used in clinics, with hundreds more registered for clinical trials. There is a significant ongoing effort to develop new tubulin-targeting chemotherapeutics, with considerable attention focused on the colchicinebinding site. This site is located at the tubulin intradimer interface and accommodates relatively simple chemical structures, such as the long-known molecule colchicine, podophyllotoxin and numerous other ligands. Previous studies have designed and characterized probes for various binding sites on tubulin. For example, several research groups have developed fluorescent derivatives of taxanes, Chatterjee et al. synthesized a fluorescently labeled vinblastine; Menchon et al. created a fluorescent analog of maytansine; Tahir et al. developed a [ 3 H]colchicine competitionbinding scintillation proximity assay; several other groups have attached fluorescent labels to colchicine, colcemid, or identified other fluorescent colchicine site binders. Each of these molecules, in principle, enables a screening of potential ligands for tubulin binding at a given specific site, as determined by competition with the fluorescent probe. However, no probe has so far been reported to sense or discriminate binders of more than a single binding site.