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The aim of the study was to establish a rapid and reliable method for screening CSF1R (Colony Stimulating factor 1 Receptor) signaling pathway inhibitors. Plasmid containing human CSF1R gene was constructed and transfected into U2OS cells stably expressing GFP-STAT1 (green fluorescent protein-signal transducers and activators of transcription) fusion protein. A cell line stably expressing both CSF1R and GFP-STAT1 fusion protein was generated, and named CSF1R/GFPSTAT1_U2OS. The CSF1R was expressed and confirmed to be functional, as exposure of rhM-CSF to the cells induce significant GFP-STAT1 nuclear translocation. The screening protocol for CSF1R inhibitors was then optimized basin on this. According to the results, 30 min of rhM-CSF exposure induce maximum protein nuclear translocation, and the EC50 value for rhM-CSF to induce GFP-STAT1 nuclear translocation was 43.50 ± 3.68 ng/mL, and the maximum STAT1 nuclear translocation was induced by about 300 ng/mL of rhM-CSF. The mean z’ factor of the assay was 0.77, confirming the robustness and reliability of current method. Three known CSF1R inhibitors, GW2580, Sunitinib or Imatinib mesylate concentration dependently blocked the rhM-CSF induced GFP-STAT1 nuclear translocation. Using the established assay, 81 compounds were screened, and 4 compounds emerge as positive hits. In conclusion, a U2OS cell line stably expressing CSF1R and GFP-STAT1 was generated, which can be used in screening and evaluating of novel CSF1R signaling pathway inhibitors by simply monitoring the nuclear translocation behavior of GFP-STAT1.