Non-viral gene therapy approaches have strongly established the utility of peptides as integral constituents of delivery platforms devised for efficient transfer of therapeutic molecules into cells. Among these, cell-penetrating peptides (CPPs), encompassing a family of short peptide sequences and their chimeric derivatives, have gained versatility through the addition of de novo peptide ligands primarily to facilitate cell-specific nucleic acid delivery in vitro and in vivo. The review illustrates the structural requirements of a noteworthy peptide TAT-PTD and other derivatives chiefly to exemplify their implication in gene therapy. An overview of the emerging concept and recent explorations will be presented through unique examples which form a facet in nanotechnology-based cancer therapy. Finally the basis for the utility of CPPs in plants will be discussed in view of its biotechnological potential.