Heat shock protein 90 (Hsp90) is a critical molecular chaperone protein that regulates the folding, maturation, and stability of a wide variety of proteins. In recent years, the development of Hsp90-directed inhibitors has grown rapidly, and many of these inhibitors have entered clinical trials. In parallel, the functional dissection of the Hsp90 chaperone machinery has highlighted the activity disruption of Hsp90 co-chaperone as a potential target. With the roles of Hsp90 co-chaperones being elucidated, cell division cycle 37 (Cdc37), a ubiquitous co-chaperone of Hsp90 that directs the selective client proteins into the Hsp90 chaperone cycle, shows great promise. Moreover, the Hsp90-Cdc37-client interaction contributes to the regulation of cellular response and cellular growth and is more essential to tumor tissues than normal tissues. Herein, we discuss the current understanding of the clients of Hsp90-Cdc37, the interaction of Hsp90-Cdc37-client protein, and the therapeutic possibilities of targeting Hsp90-Cdc37-client protein interaction as a strategy to inhibit Hsp90 chaperone machinery to present new insights on alternative ways of inhibiting Hsp90 chaperone machinery.