Due to the therapeutic potential of targeting protein-protein interactions (PPIs) there is a need for easily executed assays to perform high throughput screening (HTS) of inhibitors. We have developed and optimized an innovative and robust fluorescence-based assay for detecting PPI inhibitors， called FluorIA (Fluorescence-based protein-protein Interaction Assay). Targeting the PPI of RAD52 with replication protein A (RPA) was used as an example， and the FluorIA protocol design， optimization and successful application to HTS of large chemical libraries are described. Here enhanced green fluorescent protein (EGFP)-tagged RAD52 detected the PPI using full-length RPA heterotrimer coated， black microtiter plates and loss in fluorescence intensity identified small molecule inhibitors (SMIs) that displaced the EGFP-tagged RAD52. The FluorIA design and protocol can be adapted and applied to detect PPIs for other protein systems. This should push forward efforts to develop targeted therapeutics against protein complexes in pathological processes.