The field of gene therapy is starting to move towards clinical applications but is currently limited by the lack of efficient delivery systems. Cell-penetrating peptides provide a means of cellular delivery for gene therapy applications as well as delivery of traditional drugs. Using cell-penetrating peptides a range of different cargos have been successfully delivered into a number of cell types, in vitro as well as in vivo. In this review we discuss uptake mechanisms of different cell-penetrating peptides, with or without cargo. The transition from in vitro to in vivoapplications and strategies to increase the bioavailability of cell-penetrating peptides are also discussed.