Monastrol is primarily used in biological research as a cell-permeable small molecule that specifically inhibits mitosis by targeting the mitotic kinesin Eg5 (kinesin-5). This inhibition prevents the proper formation of the bipolar spindle during cell division, leading to cell cycle arrest in the prometaphase. Due to this mechanism, Monastrol serves as a valuable tool for studying mitotic processes, spindle dynamics, and kinesin motor protein functions. It is widely applied in cancer research to explore the role of Eg5 in tumor cell proliferation and as a model compound for developing anti-mitotic drugs. Unlike traditional microtubule-targeting agents such as taxanes or vinca alkaloids, Monastrol does not disrupt microtubule polymerization, making it a more selective and less toxic alternative for probing mitotic mechanisms. Its use has contributed to the development of other kinesin inhibitors with potential therapeutic applications in oncology.