Background: Structural homology modeling supported by bioinformatics analysis plays a key role in uncovering new molecular interactions within gene regulatory networks. Here, we have applied this powerful approach to analyze the molecular interactions orchestrating death receptor signaling networks. In particular, we focused on the molecular mechanisms of CD95-mediated NF-κB activation and the role of c-FLIP/NEMO interaction in the induction of this pathway. Results: To this end, we have created the homology model of the c-FLIP/NEMO complex using the reported structure of the v-FLIP/NEMO complex, and rationally designed peptides targeting this complex. The designed peptides were based on the NEMO structure. Strikingly, the experimental in vitro validation demonstrated that the best inhibitory effects on CD95-mediated NF-κB activation are exhibited by the NEMO-derived peptides with the substitution D242Y of NEMO. Furthermore, we have assumed that the c-FLIP/NEMO complex is recruited to the DED filaments formed upon CD95 activation and validated this assumption in silico. Further insight into the function of c-FLIP/NEMO complex was provided by the analysis of evolutionary conservation of interacting regions which demonstrated that this interaction is common in distinct mammalian species. Conclusions: Taken together, using a combination of bioinformatics and experimental approaches we obtained new insights into CD95-mediated NF-κB activation, providing manifold possibilities for targeting the death receptor network.