Abstract：Abstract Objective To screen novel FKBP52 inhibitors, and develop a high-throughput screening (HTS) assay. Methods The human FKBP52 gene was cloned by RT-PCR from human peripheral blood lymphocyte and recombinantly expressed in E. coli. Recombinant human FKBP52 (rhFKBP52) was purified by Ni2+-chelating affinity chromatography column. The screening assay was established using Suc-Ala-Leu-Pro-Phe-pNA as colorimetric substrate. 31,558 microbial metabolite extracts were screened. Results Recombinant expression strain BL21 (rosetta) /pET-30a/FKBP52 was successfully constructed. The purity of rhFKBP52 was over 90% by SDS-PAGE analysis. And the concentration was 0.862mg/mL. FKBP52 inhibitor FK506 showed an IC50 value of 12.035ng/mL. One hundrund and twenty-one samples were demonstrated to have high inhibitory activities against FKBP52. Further analysis by HPLC-MS, among these positive samples, eleven actinomycete extracts were proved to contain FK506, six actinomycete extracts contain FK520, and another seven actinomycete extracts contain rapamycin. Based on 100 96-plate screening data, the assay produced the signal/background ratio of 2.35±0.26, the average of Z' factors was 0.78±0.1. Conclusion This HTS assay for rhFKBP52 inhibitor is robust and sensitive, and this screening assay is not only adaptable for novel rhFKBP52 inhibitors but also for the microbial producing strains of known FKBP52 inhibitors, such as FK506, FK520 and rapamycin.