The crystal structure of BTLA/HVEM complex shows specific details of the proteins interaction. BTLA binds to a fragment of HVEM(26–33) which is located in the Cysteine Rich Domain 1 (CRD1) . We used that information to design and characterize peptide-based inhibitors of BTLA/HVEM complex formation. We confirmed that the short fragment of HVEM(23–39) binds to BTLA and blocks the BTLA/HVEM interactions. Further evaluation of individual contribution of each residue from the HVEM(23–39) fragment by a single alanine substitution approach showed that cysteine residues have a key role in the binding to BTLA. Finally, we showed that free cysteine amino acid can disrupt the BTLA/HVEM complex formation, highlighting a cysteine-related artefact in vitro. Overall, these results might be helpful for the design of new compounds targeting BTLA protein.