This article describes the development of a simple and robust fluorescence polarization (FP)-based binding assay and adaptation to high-throughput identification of small molecules blocking dsRNA binding to NS1A protein (nonstructural protein 1 from type A influenza strains). This homogeneous assay employs fluorescein-labeled 16-mer dsRNA and full-length NS1A protein tagged with glutathione S-transferase to monitor the changes in FP and fluorescence intensity simultaneously. The assay was optimized for high-throughput screening in a 384-well format and achieved a z' score greater than 0.7. Its feasibility for high-throughput screening was demonstrated using the National Institutes of Health clinical collection. Six of 446 small molecules were identified as possible ligands in an initial screening. A series of validation tests confirmed epigallocatechine gallate (EGCG) to be active in the submicromolar range. A mechanism of EGCG inhibition involving interaction with the dsRNA-binding motif of NS1A, including Arg38, was proposed. This structural information is anticipated to provide a useful basis for the modeling of antiflu therapeutic reagents. Overall, the FP-based binding assay demonstrated its superior capability for simple, rapid, inexpensive, and robust identification of NS1A inhibitors and validation of their activity targeting NS1A.