HSP90-CDC37 protein-protein interaction (PPI) works as a kinase specific-molecular chaperone system to regulate the maturation of kinases. Currently, selectively disrupting HSP90-CDC37 PPI, rather than the direct inhibition of the ATPase function of HSP90, is emerging as a promising strategy for cancer therapy by specifically blocking the maturation of kinases. However, due to the limited understanding of HSP90-CDC37 binding interface, design of small molecule inhibitors targeting HSP90-CDC37 PPI is challenging. In this work, based on the binding mode of compound 11 (previously reported by our group), we discovered a hydrophobic pocket centered on Phe213, which was previously unknown, contributing to the binding affinity of HSP90-CDC37 PPI inhibitors. A series of hydrophobic substituted inhibitors were utilized to confirm the importance of Phe213 hydrophobic core. Finally, we obtained an optimum compound DDO-5994 (exhibited an ideal binding pattern on hydrophobic core) with improved binding affinity (K_D = 5.52 μM) and antiproliferative activity (IC₅₀ = 6.34 μM). Both in vitro and in vivo assays confirmed DDO-5994 as a promising inhibitor to exhibit ideal antitumor efficacy through blocking HSP90-CDC37 PPI.