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In drug discovery research, the selection of promising binding sites and understanding the binding mode of compounds are crucial fundamental studies. The current understanding of the proteins-ligand binding model extends beyond the simple lock and key model to include the induced-fit model, which alters the conformation to match the shape of the ligand, and the pre-existing equilibrium model, selectively binding structures with high binding affinity from a diverse ensemble of proteins. Although methods for detecting target protein binding sites and virtual screening techniques using docking simulation are well-established, with numerous studies reported, they only consider a very limited number of structures in the diverse ensemble of proteins, as these methods are applied to a single structure. Molecular dynamics (MD) simulation is a method for predicting protein dynamics and can detect potential ensembles of protein binding sites and hidden sites unobservable in a single-point structure. In this study, to demonstrate the utility of virtual screening with protein dynamics, MD simulations were performed on Trypanosoma cruzi spermidine synthase to obtain an ensemble of dominant binding sites with a high probability of existence. The structure of the binding site obtained through MD simulation revealed pockets in addition to the active site that was present in the initial structure. Using the obtained binding site structures, virtual screening of 4.8 million compounds by docking simulation, in vitro assays, and X-ray analysis was conducted, successfully identifying two hit compounds., Competing Interests: The authors declare the following competing financial interest(s): Y.H., Y.A., Y.T., K.O., I.N., T.N., and M.O. were employees of Astellas Pharma Inc. at the time this study was being conducted. The company had no role in the design of the study, namely in the collection, analysis, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. R.Y., N.Y., T.I., D.K.I., K.K., Y.A., and M.S. declare that they have no conflict of interest., (© 2023 The Authors. Published by American Chemical Society.)