Cell-penetrating peptide based vehicles have been developed for the delivery of different payloads into the cells in culture and in animals. However, several biological features, among which is the tendency to trigger innate immune response, limit the development of highly efficient peptide-based drug delivery vectors. This study aims to evaluate the influence of transportan 10 (TP10) and its chemically modified derivatives, PepFects (PFs), on the innate immune response of the host system. PFs have shown high efficiency in nucleic acid delivery in vitro and in vivo; hence, the estimation of their possible toxic side effects would be of particular interest. In this study, we analyzed cytotoxic and immunogenic response of PF3, PF4, and PF6 peptides in monocytic leukemia and peripheral blood mononuclear cell lines. In comparison with amphipathic PFs, TP10, TAT, stearyl-(RxR)(4) peptides, and the most widely used transfection reagents Lipofectamine 2000 and Lipofectamine RNAiMAX were also analyzed in this study. IL-1β, IL-18, and TNF-α cytokine release was detected using highly sensitive enzyme-linked immunosorbent assay (ELISA). Cell viability was detected by measuring the activity of cellular enzymes that reduce water-soluble tetrazolium salts to formazan dyes and apoptosis was evaluated by measuring the levels of caspase-1 and caspase-3/7 over untreated cells. All peptides were found to be nontoxic and nonimmunogenic in vitro at the concentrations of 10 μM and 5 μM, respectively, and at a dose of 5 mg/kg in vivo, suggesting that these CPPs exhibit a promising potential in the delivery of therapeutic molecules into the cell without risks of toxicity and inflammatory reactions.