The selective permeability of the plasma membrane prohibits most exogenous agents from gaining cellular access. Since many therapeutics and reporter molecules must be internalized for activity, crossing the plasma membrane is essential. A very effective class of transporters harnessed for this purpose are cell penetrating peptides (CPPs), a group of short cationic sequences with a remarkable capacity for membrane translocation. Since their discovery in 1988, CPPs have been employed for the delivery of a wide variety of cargo including small molecules, nucleic acids, antibodies and nanoparticles. This review describes recent advances in the use of CPPs for biological and therapeutic applications. In particular, an emphasis is placed on novel systems and insights acquired since 2006. Basic research on CPPs has recently yielded techniques that provide further information on the controversial mechanism of CPP uptake and has also resulted in the development of new model membrane systems to evaluate these mechanisms. In addition, recent use of CPPs for the development of new cellular imaging tools, biosensors, or biomolecular delivery systems have been highlighted. Lastly, novel peptide delivery vectors, designed to tackle some of the drawbacks of CPPs and enhance their versatility, will be described. This review will illustrate the diverse applications for which CPPs have been harnessed and also demonstrate the remarkable advancements these peptides have facilitated in cell biology.