The aim of this study was to identify novel TNF-alpha blocking peptide(s) using phage display technology. Two novel 7-mer TNF-alpha binding peptides P51 and P52 with Kd values of 1.47 and 0.51 nM were identified. Phage particles displaying P51 and P52 peptides at 0.318 nM concentration prevent cytotoxic effect of TNF-alpha on L929 cells by 8.2% and 16.15%, respectively. Synthesized P51 and P52 peptides also inhibited TNF-alpha induced cytotoxicity with IC50 values of 25.15 +/- 2.18 and 7.08 +/- 2.24 mu M, respectively. The result of RT-PCR also supports the inhibitory activity of the identified peptides, where P51 and P52 significantly inhibit the inductive effect of TNF-alpha on I kappa B-alpha mRNA levels. The inhibitory effects of the peptides were attributed to their abilities of binding at the intersubunit interfaces leading to TNF-alpha dissociation. The results of molecular docking studies revealed that the peptides- TNF-alpha complexes are mostly stabilized by hydrophobic contacts. (C) 2016 Elsevier B. V. All rights reserved.