Objective:Taking PDCD10 (homo sapiens programmed cell death 10) and MST4 (Mst3 and SOK1-related kinase) as drug targets, to establish a low molecular weight inhibitors screening method in vitro. Methods:The open reading frames of PDCD10 and MST4 were reconstructed into the prokaryotic expressive vector pGEX4T-2. The recombinant proteins were purified using GST affinity chromatograph, and then analyzed using Western blot. ELISA assay was utilized to detect the activity of purified proteins of GST-PDCD10 and GST-MST4 and screen for the inhibitor in 825 compounds, which were verified by the dual luciferase reporter gene assay in Elk1 signaling pathway. Results:Drug targets PDCD10 and MST4 inhibitors screening method was established successfully in vitro. One compound screened by this method was confirmed to inhibit the activity of PDCD10 and MST4. Conclusion: The method is of better parallelism and stability. The screened compound could also inhibit the activity of PDCD10 and MST4 in Elk1 signaling pathway.