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Cell penetrating peptides (CPPs) are peptides with the capacity to translocate across the plasma membrane of mammalian cells. These biological macromolecules, have created a new horizon in biomedical research to obtain most efficient ways to carry substantial cargoes such as peptides, proteins, plasmid DNA, oligonucleotides, peptide nucleic acids (PNA), and even nanoparticles or liposomes across the plasma membrane into cellular compartments. Circular dichroism allows a quick estimation of secondary structures of cell-penetrating peptides and is well suited for following changes in their secondary structures dependent on peptide concentration, pH, buffer, and on the nature and composition of the lipid.