The p53-MDM2 interaction is a well-known protein-protein contact, and its disruption is a key event for p53 activation and induction of its oncosuppressor response. The design of small molecules that can block the p53-MDM2 interaction and reactivate the p53 function is a promising strategy for cancer therapy. To date, several compounds have been identified as p53-MDM2 inhibitors, and X-ray structures of MDM2 complexed with several ligands are available in the Brookhaven Protein Data Bank. These data have been exploited to compile a hierarchical virtual screening protocol. The first steps were aimed at selecting a focused library, which was submitted in parallel to docking and pharmacophore model alignment. Selected compounds were subjected to inhibition assays of both cellular vitality (MTT) and p53-MDM2 interaction (ELISA and co-immunoprecipitation), disclosing four nanomolar inhibitors.