Hsp90 is an evolutionarily conserved molecular chaperone that modulates many cellular processes in eukaryotes through signaling pathways regulated by Hsp90-related proteins such as proliferation, cell cycle, and gene expression. Thanks to control of the Hsp90 protein, cellular functions such as cell proliferation and apoptosis are stabilized [1,2]. In addition, Hsp90 inhibitors have a wide therapeutic window for cancer therapy, since the level of Hsp90 between cancer cells and normal cells is distinctive. Therefore, targeting Hsp90 today is a promising strategy for developing a new anticancer drug [3]. For this purpose, the interactions of all drugs (FDA approved, experimental, research) with Hsp90 protein in the Drugbank database were determined using structure-based virtual screening. In this study, all molecules (~10767) in the Drugbank database were downloaded and examined for interactions with the Hsp90 protein and potential hit inhibitors were identified. DB00157, DB00581, DB00256, DB0786, DB00577, DB00415 targeting the active site of Hsp90 were identified by virtual screening and molecular dynamics simulations were performed. After MD simulation (200 ns), the effects of NADH, Lactulose and Ampicillin (DB00157, DB00581 and DB00415) drugs on human breast cancer cell lines (MCF-7 and MCF10) were determined as in vitro. IC50 values of 17-AAG, Ampicillin, Lactulose and NADH applied (48 hours) on MCF-7 and MCF10A cell lines were determined experimentally. It was determined that three of these six target drugs (Lactulose, NADH and Ampicillin) determined by in silico modeling according to their IC50 values, acted against MCF-7 and MCF10A human breast cancer cell lines at moderate micromolar concentrations. The antiproliferative effects of Lactulose, Ampicillin, NADH and reference 17-AAG drugs were evaluated in MCF-7 breast cancer and MCF10A cells after prolonged incubation with increasing compound concentrations. These results accelerated their identification as new Hsp90 inhibitors that could be used in cancer therapy. Considering the ligand RMSD values obtained as a result of molecular dynamic simulation studies of these target compounds, new Hsp90 target compounds can be designed by removing the groups with high fluctuations on the ligand.