Aim: To develop a homogeneous high-throughput screening (HTS) assay based on scintillation proximity assay (SPA) technology for identification of novel α4β2 nicotinic acetylcholine receptor (nAChR) modulators. Methods: Membrane preparation of HEK293 cells expressing α4β2 nAChR, [3~H]cytisine and wheat germ agglutinin (WGA)-coupled microbeads were used to develop an HTS assay based on SPA technology. This method was validated against a conventional filter binding approach and applied to large-scale screening of a library containing 32000 synthetic compounds. Intracellular calcium measurement was carried out to verify the bioactivities of the hits found by the SPA assay. Results: IC_(50) values of 2 reference compounds (epibatidine and RJR 2403) determined by SPA and filter binding methods were comparable and consistent with those reported elsewhere. A total of 54 compounds, showing more than 60% competitive inhibition on [3~H]cytisine binding to α4β2 nAChR, were identified initially following an HTS campaign. Secondary screening confirmed that 17 compounds with novel chemical structures possessed relatively high binding affinity to α4β2 nAChR (K_i<2 μmol/L). Eight compounds displayed antagonistic effects with>50% inhibition on ABT-594-induced calcium mobilization while none showed any agonistactivity. Conclusions: This homogeneous binding assay is a highly efficient, amenable to automation and robust tool to screen potential α4β2 nAChR modulators in an HTS setting. Its application may be expanded to other membrane receptors and ion channels. Aim: To develop a homogeneous high-throughput screening (HTS) assay based on scintillation proximity assay (SPA) technology for identification of novel α4β2 nicotinic acetylcholine receptor (nAChR) modulators. Methods: Membrane preparation of HEK293 cells expressing α4β2 nAChR, H] cytisine and wheat germ agglutinin (WGA) -coupled microbeads were used to develop an HTS assay based on SPA technology. This method was validated against a conventional filter binding approach and applied to large-scale screening of a library containing 32000 synthetic compounds. Intracellular calcium measurement was carried out to verify the bioactivities of the hits found by the SPA assay. Results: IC 50 values ​​of 2 reference compounds (epibatidine and RJR 2403) determined by SPA and filter binding methods were comparable and consistent with those reported A total of 54 compounds, showing more than 60% competitive inhibition on [3 ~ H] cytisine binding to α4β2 nAChR, were identified initially Secondary compounds confirmed antagonistic effects with> 50% inhibition on ABT-594-induced calcium (K_i <2 μmol / L) following an HTS campaign. mobilization while none showed any agonist activity. Conclusions: This homogeneous binding assay is a highly efficient, amenable to automation and robust tool to screen potential α4β2 nAChR modulators in an HTS setting. Its application may be expanded to other membrane receptors and ion channels.