The introduction of new anti-HCV drugs in the therapy is an imperative need and is necessary with a view to develop an interferon-free therapy. Thus, the discovery and the development of novel small molecule inhibitors of the viral NS5B polymerase represent an exciting area of research for many pharmaceutical companies and academic groups. This study represents a contribution to this field, and relies on the identification of the best NS5B model(s) to be used in structure-based computational approaches aimed at identifying novel non nucleoside inhibitors of one of the protein allosteric sites, namely palm site I. Firstly, the NS5B inhibitors at palm site I were classified as Water-Mediated or Non Water-Mediated ligands depending on their ability to interact with or displace a specific water molecule. Then, we took advantage of the available X-ray structures of NS5B/ligand complexes to build different models of protein/water combinations, which were used to investigate the influence on docking studies of solvent sites as well as of the influence of the protein conformations. As overall trend, we observed improved performance in the docking results of Water-Mediated Inhibitors by inclusion of explicit water molecules, with an opposite behavior generally happening for the Non Water-Mediated Inhibitors. The best performing target structures for the two ligand sets were then used for virtual screening simulations of a library containing the known NS5B inhibitors along with related decoys to assess the best performing targets ensembles on the basis of their ability to discriminate active and inactive compounds as well as to generate the correct binding modes. The parallel use of different protein structures/water sets outperformed the use of a single target structure, with the two-protein 3H98/2W-2FVC/7W and 3HKY/NoW-3SKE/NoW models resulting the best performing ensembles for Water-Mediated Inhibitors and Non Water-Mediated Inhibitors, respectively. The information gathered from this work confirms the primary role of water molecules and protein flexibility in docking-based studies, and can be exploited to aid NS5B-directed HCV drug discovery efforts.