Aim:To develop a complex high-throughput screening (HTS) assay based onscintillation proximity assay (SPA) technology for identification of novel peroxi-some proliferator-activated receptor gamma (PPARγ) modulators.Methods:Full-length PPARγ and retinoid X receptor alpha (RXRα),biotinylated PPAR responseelement (PPRE),[~3H]BRL49653 and streptavidin-coated FlashPlate or microbeadwere used to develop an HTS assay based on SPA technology.This‘ABCDE’method was validated against conventional hydroxyapatite (HA) assay and ap-plied to large-scale screening of 16 000 synthetic compounds and natural productextracts.Results:(1) IC_(50) values of positive control compounds (BRL49653 andtroglitazone) obtained from the‘ABCDE’method and HA assay were compa-rable and consistent with those reported elsewhere;(2) Approximately 178compounds,showing more than 70% competitive inhibition on BRL49653 bind-ing to PPARγ,were identified initially by the‘ABCDE’method (microbead);(3)Secondary screening using FlashPlate and cross-reactivity studies with RARα,β,γ and RXRα,β,γ confirmed that 12 compounds possessed specific PPARγ bind-ing properties including 2 with IC_(50) values less than 0.5 μmol/L and novel chemi-cal structures.Conclusions:The‘ABCDE’method using either FlashPlate ormicrobead,is a highly efficient,automatable,and robust tool to screen potentialPPARγ modulators in HTS setting.Its application may be expanded to othernuclear receptors that form heterodimers upon activation. Aim: To develop a complex high-throughput screening (HTS) assay based on scintillation proximity assay (SPA) technology for identification of novel peroxi-some proliferator-activated receptor gamma (PPARγ) modulators. Methods: Full- length PPARγ and retinoid X receptor alpha (RXRα), biotinylated PPAR response element (PPRE), [~ 3H] BRL49653 and streptavidin-coated FlashPlate or microbeadwere used to develop an HTS assay based on SPA technology. This’ABCDE’method was validated against conventional hydroxyapatite (HA) assay and ap -plied to large-scale screening of 16 000 synthetic compounds and natural productextracts. Results: (1) IC 50 values ​​of positive control compounds (BRL49653 andtroglitazone) obtained from the’ABCDE’method and HA assay were compa-rable and consistent (2) Approximately 178 compounds, showing more than 70% competitive inhibition on BRL49653 bind-ing to PPARγ, were identified initially by the ’ABCDE’method (microbead); (3) Secondary scre ening using FlashPlate and cross-reactivity studies with RARα, β, γ and RXRα, β, γ confirmed that 12 compounds possessed specific PPARγ bind-ing properties including 2 with IC 50 values ​​less than 0.5 μmol / L and a novel chemi-cal structures.Conclusions: The’ABCDE’method using either FlashPlate ormicrobead, is a highly efficient, automatable, and robust tool to screen potential PPARγ modulators in HTS setting. Applications may be expanded to other nuclear receptors that form heterodimers upon activation.