Signal transducer and activator of transcription 3 (STAT3) is overactive in a wide variety of human tumors. Activity of STAT3 requires its own SH2-domain-mediated binding to phosphotyrosine-containing sequences. We have developed a high-throughput binding assay, based on fluorescence polarization, which allows screening for small molecules that bind to the STAT3 SH2 domain and thereby inhibit its activity. The basis of this assay is the binding of a fluorescein-labeled phosphotyrosine-peptide derived from the interleukin-6 receptor subunit gp130 to unphosphorylated STAT3 with a K(d) of 150 nM. The assay is stable with regard to salt concentration, dimethyl sulfoxide concentration, and time. It has been adapted to a 384-well format, with a Z' value of 0.87, and can be used to screen for small molecules that bind to the STAT3 SH2 domain.