TNFα inhibitors have been successfully developed and used in the clinical treatment of autoimmune disorders. However, the use of TNFα blockade may be accompanied by side effects. The cases of bacterial and viral infections, lymphoproliferative disorders, and anti-TNFα-induced lupus, have been reported among the rheumatoid arthritis or Crohn's disease patients treated with TNFα blockers. Therefore, alternative therapeutic strategy is highly desirable. TNFα signaling via TNFR1 induces proinflammatory responses, and selective inhibition of TNFR1 signaling may be beneficial for managing autoimmune diseases. To this end, we developed minibinder proteins targeting soluble ectodomain of TNFR1 (sTNFR1) by de novo computational designing. Top-rated designed minibinders targeting to two hydrophobic sites on sTNFR1 were selected and expressed in E. coli. Purified top-ranked minibinders are thermostable in solution and presented micromolar to sub-micromolar affinity to sTNFR1. All designs showed the potency of blocking TNFα signaling in L929 cell-based assays, and one of the designs targeting to hydrophobic Site I called S1B2 exhibited higher potency than other minibinders with IC