Two homogeneous high-throughput assays, AlphaScreen and fluorescence polarization, were established to quantify inhibitor selectivity between different protein-protein complexes. As a first case study, they have been successfully applied to the key protein-protein interactions in the downstream sites of the canonical Wnt signaling pathway. The aberrant formation of the β-catenin/T-cell factor (Tcf) complex is the major driving force for many cancers and fibroses. Crystallographic and biochemical studies reveal that the binding modes of Tcf, E-cadherin, and adenomatous polyposis coli (APC) to β-catenin are identical and mutually exclusive. In the present study, two highly sensitive and robust assays were established to quantitatively evaluate inhibitor selectivity between β-catenin/Tcf, β-catenin/E-cadherin, and β-catenin/APC interactions. A pilot screen demonstrated the feasibility of the assays and yielded four hits for the disruption of β-catenin/Tcf interactions. A potent and dual-selective β-catenin/Tcf inhibitor was identified.