The molecular chaperone Hsp90 is overexpressed in prostate cancer (PCa) and is responsible for folding, stabilisation and maturation of many oncoproteins implicated in PCa progression. Consequently, targeting Hsp90 by small molecule inhibitors is a rational strategy for treatment of advanced PCa. Unfortunately, while agents such as 17-allylamino-demethoxygeldanamycin (17AAG) and AUY922 have demonstrated promising efficacy in cell lines, animal models, and tumour tissues cultured as explants, these Hsp90 inhibitors, currently undergoing clinical trials, also induce a heat shock response (HSR) in target cells. This leads to accumulation of various heat shock proteins, notably Hsp27 and Hsp70, which have cytoprotective properties and may represent an important mechanism of clinical resistance to these agents. Our research has resulted in the development of a new class of Hsp90 inhibitors that target a different domain of Hsp90 compared to previous Hsp90 inhibitor compounds and do not induce HSR. In this study we demonstrated that two promising new Hsp90 inhibitors, SM253 and SM258, do not result in elevated expression of Hsp27 or Hsp70. This was revealed by qPCR and Western blot of PCa cell lines (22Rv1, LNCaP and PC3) after 48hrs culture with DMSO (vehicle control), 17-AAG (50nM), AUY922 (25nM), SM253 (5uM), or SM258 (5uM). Furthermore, cleaved caspase-3 staining in cell lines and tumour tissues cultured as explants clearly demonstrated these novel inhibitors are capable of significantly inducing apoptosis of PCa cells at low micromolar concentrations. The efficacy of SM253 and SM258 treatment in PCa cell lines and explant tissues earmarks this new class of inhibitors for further clinical evaluation, particularly as they offer a novel strategy to target Hsp90 without inducing protein pathways implicated in drug resistance. Ultimately, this study indicates that the design and use of alternate Hsp90 inhibitors will maintain a focus on Hsp90 as a highly promising oncogenic target for PCa treatment.