The Hippo signaling pathway controls cell-cell contact, cell proliferation, as well as organ size by integrating changes in the cellular microenvironment. In recent years, the pivotal role of Hippo signaling in cancers has been well recognized. Inhibition of the pathway promotes the translocation of the major Hippo pathway effectors, the yes-associated protein (YAP) and its paralog TAZ, to the nucleus, where they interact with the transcription factor family transcriptional enhancer associate domain (TEAD), thus coactivating the expression of downstream genes, leading to cell transformation, tissue overgrowth, and tumor development. Therefore, the interruption of the YAP-TEAD transcriptional complex represents a novel opportunity for the treatment of cancer. Here, we established a fluorescence polarization (FP)-based assay for the identification and evaluation of YAP-TEAD protein-protein interface (PPI) inhibitors at the YAP Ω-loop binding region of TEAD, which is also called interface 3 at the YAP-TEAD binding surface. Furthermore, a patented small molecule (Patent-22) was evaluated by the FP assay, which confirmed that it was a YAP-TEAD PPI inhibitor at interface 3. Possessing great application value, this FP method is reliable, robust, and economical for inhibitor assessment and drug discovery.