Aim:To develop and optimize a competitive fluorescence polarization (FP) method, and use it as a high-throughput screening (HTS) assay for drug discovery.Methods:Human lectin-1ike oxidized low-density lipoprotein receptor-1 (hLOX-1) and oxidized low-density lipoprotein (oxLDL) were used to establish a high-throughput fluorescence polarization assay to screen ligands of human LOX-1.A 96-well plate assay was performed with a fast plate reader.Three fluorescein isothiocyanate.1abeled hLOX-1 concentrations (100,200,and 400 nmol/L) were selected to be titrated by oxLDL (from 0.05 nmol/L to 100μmol/L) in order to obtain optimal reactive concentrations.The concentration of Me2SO used (0%,1%,3%,5%) andincubationtime (15min,30min,1h,2h) were optimized.The Z’ factor was calculated to estimate the quality of FP-based HTS.Results:Concentrations of 200 nmol/L for human LOX-1 and 50 μmol/L for oxLDL were used in the actual assay.Concentrations of 0% to 5% Me2SO and different reaction times did not affect the FP-based HTS.The Z′value was 0.66.By using this detection and screening system,12 700 compounds were screened and 3 ligands with an IC50 of less than 4.5μmol/L were found.Conclusion:The established competitive FP-based assay is sensitive,stable,highly reproducible and robust,and suitable for HTS for ligands of the hLOX-1 receptor.