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-like 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-labeled hLOX-1 concentrations (100, 200, and 400 nmol/L) were selected to be titrated by oxLDL (from 0.05 nmol/L to 100 μmol/L) in order to obtain optimal reactive concentrations. The concentration of MeSO used (0%, 1%, 3%, 5%) and incubation time (15 min, 30 min, 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% MeSO 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 4.5 μmol/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. ©2006 CPS and SIMM.