During interphase, the transport receptor importin-β carries cargoes into the nucleus, where RanGTP releases them. A similar mechanism operates in mitosis to generate a gradient of active spindle assembly factors around mitotic chromosomes. Importin-β and RanGTP have been implicated in additional cellular processes, but the precise roles of the Ran/importin-β pathway throughout the cell cycle remain poorly understood. We implemented a FRET-based, high-throughput small molecule screen for compounds that interfere with the interaction between RanGTP and importin-β and identified importazole, a 2,4-diaminoquinazoline. Importazole specifically blocks importin-β-mediated nuclear import both in Xenopus egg extracts and cultured cells, without disrupting transportin-mediated nuclear import or CRM1-mediated nuclear export. When added during mitosis, importazole impairs the release of an importin-β cargo FRET probe and causes both predicted and novel defects in spindle assembly. Together, these results indicate that importazole specifically inhibits the function of importin-β, likely by altering its interaction with RanGTP. Importazole is a valuable tool to evaluate the function of importin-β/RanGTP pathway at specific stages during the cell cycle.