An understanding of the dissociation of penetratin is important for improving its metabolic stability and cargo-delivery efficiency. In this study, we describe the selective cleavage of the K15–K16 amide bond of penetratin under the low-energy collision-induced dissociation condition in mass spectrometry. A variety of penetratin substitutes have been studied in which key basic amino acids have been substituted within the sequence. The calculated structure indicates that an α-helix structure prevents the fragmentation of the central peptide domain and the side chain of lysine is involved in the proton translocation process. Copyright © 2010 John Wiley & Sons, Ltd.