Virtual screening methods combined with experimental assays were used to identify low molecular weight inhibitors for Src homology 2 domain-containing phosphatase 2 (SHP-2) that is mutated and hyperactivated in Noonan syndrome and a significant portion of childhood leukemias. Virtual screening included multiple conformations of the protein, score normalization procedures, and chemical similarity considerations. As the catalytic core of SHP-2 shares extremely high homology to those of the related SHP-1 phosphatase and other tyrosine phosphatases, in order to identify selective inhibitors, we chose to target an adjacent protein surface pocket that is predicted to be important for binding to phosphopeptides and that has structural features unique to SHP-2. From a database of 1.3 million compounds, 9 out of 165 computationally selected compounds were shown to inhibit SHP-2 activity with IC(50) values of approximately 100 microM. Two of the active compounds were further verified for their ability to inhibit SHP-2-mediated cellular functions. Fluorescence titration experiments confirmed their direct binding to SHP-2. Because of their simple chemical structures, these small organic compounds have the potential to act as lead compounds for the development of novel anti-SHP-2 drugs.