Toll-like receptors (TLR), a family of closely related type I, transmembrane, signal transducing proteins, sense invading pathogens early in the immune response to infection and deliver intracellular signals to the cell. Both TLRs and their adapter proteins possess a conserved region, the Toll/IL-1 resistance (TIR) domain. A subregion of approximately 14 amino acids within the TIR domain, the BB loop, enables interactions between certain TLRs or between certain TLRs and their adapter molecules. Use of cell-penetrating decoy peptides composed of the sequence of the Drosophila antennapedia peptide (16 amino acids) juxtaposed to a specific TIR BB loop 14 amino acid sequences enables an evaluation of the relative efficacy of such BB loop peptides to inhibit TIR-TIR interactions and signaling. Moreover, failure of specific BB loop peptides to inhibit signaling suggests that this region of a particular TIR domain is likely to not be involved in signaling. This review discusses cell-penetrating decoy peptides as a new tool to further understanding of the molecular interactions required for TLR signaling and evaluates the potential of this approach for the creation of therapeutic agents.