MyD88 is an essential adaptor protein that mediates IL-1R/TLR signals. The homogeneous dimerization of MyD88 is required for its recruitment to the membrane receptors and is achieved by the interaction of its C-terminate TIR domain. After binding with the receptor, MyD88 dimer recruits the downstream molecules transmitting the inflammation signals and inducing gene expression. The present study aimed to establish a living-cell-fluorescence-based and high-through-put model for screening the inhibitors against the dimerization of MyD88 TIR. We constructed GFP-MyD88 TIR and RFP-MyD88 TIR plasmids, and transiently transfected them along with GFP or RFP into HeLa cells with different combinations. Under the excitation at 488 nm, the cells transfected with GFP-MyD88 TIR and RFP-MyD88 TIR plasmids showed the energy transfer from GFP to RFP, which is supposed to be mediated by the interaction of MyD88 TIR. Nevertheless, in the cells transfected with GFP-MyD88 TIR and RFP or RFP-MyD88 TIR and GFP plasmids, FRET was obviously impaired due to the absence of MyD88 TIR dimerization. The results suggest a possibility to establish a model for screening the inhibitors against the dimerization of MyD88 TIR: select the cell line that dually expresses GFP-MyD88 TIR and RFP-MyD88 TIR, and monitor the alteration of FRET upon the adding of different compounds in the medium. Additionally, we isolated and purified recombinant proteins His-MyD88 TIR and GST-MyD88 TIR, and performed in vitro protein binding assay. This recombinant protein binding assay can be used to verify the inhibitor candidates selected from the fluorescence screening model directly blocking the interaction of MyD88 TIR or not. The fluorescence screening model plus the in vitro verifying assay can be broadly used with commercially available compounds banks or lab-made natural products to find effective anti-inflammation inhibitors for therapeutic treatment of MyD88-pathway related chronic inflammation or autoimmune disorders.