Aim To develop an in vitro high throughput drug screening system based on reporter gene assay for identification of novel compounds with PXR, FXR and LXRα agonist activity. Methods The expressions of exogenous PXR, FXR and LXRαgene in HEK293, HepG2 and LS174T cells were examined by Real-Time quantity PCR. pSG5-hPXR and pGL3-XREM-CYP3A4, pEGFP-N3-hFXR and EcRE-TK-Luc, pCMX-FLAG-hLXRα and pGL3-XREM-CYP3A4 were cotransfected into cells and the optimal ratio of three plasmids was determined. The dose-response relationship between the positive drug and the fold induction was determined. The specificity of the model was ex-amined, and the repeatability was also determined by Z′ value. Results ① The PXR, FXR and LXRα mRNA expression in HEK293 cell is low among three different cells. ②reporter gene vector and expression plasmid ratio of 1∶ 1, 2∶ 1 and 2∶ 1 were proved to be suitable for highest relative luciferase activity for PXR, FXR or LXRα agonist screening model. ③ The relative luciferase activity was induced by Rif, CDCA or T0901317 in a dose-dependent manner. ④Only Rif, CDCA or T0901317 could significantly increase the relative luciferase activity in PXR,FXR or LXRα agonist screening model, no effect of other nuclear re-ceptors agonist was observed, and the values of Z′-factor for PXR, FXR and LXRαagonist screening model were 0. 58, 0. 66 and 0. 63, respectively. Conclusion An in vitro PXR, FXR and LXRα agonist high-throughput screening models are devel-oped with acceptable specificity and repeatability, and the mod-els can be used to screen PXR, FXR and LXRα agonist.