Kinesin-5 motor proteins are essential for mitotic spindle formation and maintenance, ensuring accurate chromosome segregation. Human kinesin-5 is highly expressed in various cancer cells but not in nonproliferative tissues; therefore, it is expected to be an attractive target for cancer chemotherapy, with fewer adverse side effects. Many inhibitors have been developed and subjected to clinical trials; however, they have not yet been commercially distributed because of their poor efficacy and frequent drug resistance. Establishing in vivo assay systems to easily monitor inhibitory activity is necessary and valuable to develop more effective inhibitors. Here, we report a procedure to evaluate the inhibitory activity against human kinesin-5 using a fission yeast-based system called “in schizo”. Our approach could further be used to screen for inhibitors against kinesin-5 and other human cancer-related targets.