Photoswitchable compounds are potent tools for elucidating molecular functions in dynamic cellular processes. Photoswitchable inhibitors targeting various mitotic spindle factors have been developed. In this chapter, we describe experimental methods for photo-controlling mitotic chromosome dynamics using a recently developed photoswitchable inhibitor of mitotic kinesin, CENP-E. This inhibitor undergoes reversible photoisomerization to a more inhibitory trans or less inhibitory cis state by visible or UV light irradiation, respectively, enabling photoswitching of CENP-E function both in vitro and in vivo. First, we explain the procedures used to optimize the experimental condition for efficient photoswitching of CENP-E functionality in cultured cells. We then describe how to conduct de novo photo-control of mitotic chromosome motion using the inhibitor under a microscope.