Abstract Phoneutria nigriventer spider can cause severe envenomation in humans principally due to its venom toxin δ-ctenitoxin-Pn2a. Current low yielding antivenom production is extremely complicated and dangerous. Furthermore, δ-ctenitoxin-Pn2a cystine-knot motif provides exceptional stability hampering immune response activation. Here, epitopes from δ-ctenitoxin-Pn2a were identified, and antigenic peptides were designed for their potential use in antivenom production. The Immune Epitope Database Analysis Resource was used to identify the G34YFWIAWYKLANCKK48 epitope and used to design antigenic peptides. The Cys was replaced by α-aminobutyric acid (Abu) to avoid disulfide bonds formation. To increase their immunogenicity, branched and N-palmitoylated peptides were synthesized. Ac-GYFWIAWYKLAN-Abu-KKG-NH2 (A), (Ac-GYFWIAWYKLAN-Abu-KK)2-KG-NH2 (B), Palm-GYFWIAWYKLAN-Abu-KKG-NH2 (C) and (Palm-GYFWIAWYKLAN-Abu-KK)2-KG-NH2 (D) were synthesized using solid-phase peptide synthesis (SPPS) techniques and analyzed by ESI-MS demonstrating their identity. Also, they were evaluated by RP-HPLC, and all the chromatograms showed only one principal peak except that of the N-palmitoylated branched peptide which showed two principal peaks probably due to the presence of two conformations in slow interconversion. Cytotoxicity was evaluated on the murine macrophage cell line RAW264.7 by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay in the presence of increasing doses of each peptide (0.25-10.0 µM). Peptide A did not exhibit cytotoxicity between 0.25-10.0 µM, while B, C and D showed cytotoxicity over 10.0, 5.0 and 2.5 µM respectively. NF-κB cellular distribution was evaluated by immunofluorescence, after exposing macrophages to 0.5 µM of each peptide. An early activation was observed for all the assayed peptides demonstrating that they are promising candidates for their in vivo evaluation as immunogens in antivenom production.