Hsp90 has emerged as an important anti-cancer drug target because of its essential role in promoting the folding and maturation of many oncogenic proteins. Here we describe the development of the first high throughput screen, based on AlphaScreen™ technology, to identify a novel type of Hsp90 inhibitors that interrupt its interaction with the cochaperone HOP. The assay uses the 20-mer C-terminal peptide of Hsp90 and the TPR2A domain of HOP. Assay specificity was demonstrated by measuring different interactions using synthetic peptides, with measured IC50s in good agreement with reported values. The assay is stable over 12 hours and tolerates DMSO up to 5%. We first validated the assay by screening against 20,000 compounds in 384-well format. After further optimization into a 1536-well format, it was screened against a NCGC library of 76,134 compounds, with a signal-to-background (S/B) ratio of 78 and Z’ factor of 0.77. The present assay can be used for discovery of novel small molecule Hsp90 inhibitors that can be used as chemical probes to investigate the role of cochaperones in Hsp90 function. Such molecules have the potential to be developed into novel anti-cancer drugs, for use alone or in combination with other Hsp90 inhibitors.