The abnormal activity of Fyn tyrosine kinase has been shown to be related to various human cancers. Furthermore, its involvement in signaling pathways that lead to severe pathologies, such as Alzheimer's and Parkinson's diseases, has also been demonstrated, thus making Fyn an attractive target for the discovery of potential novel therapeutics for brain pathologies and tumors. In this study we evaluated the reliability of various screening approaches based on the FLAP software. By the application of the best procedure, the virtual screening workflow was used to filter the Gold and Platinum database from Asinex to identify new Fyn inhibitors. Enzymatic assays revealed that among the eight top-scoring compounds five proved to efficiently inhibit Fyn activity with IC50 values in the micromolar range. These results demonstrate the validity of the methodologies we followed. Furthermore, the five active compounds herein described may be considered as interesting leads for the development of new and more efficient Fyn inhibitors.