Cell penetrating peptides (CPPs) belong to the large family of membrane active peptides that comprises antimicrobial and viral fusion peptides with whom they share many properties. CPPs have been increasingly used to transport a wide range of molecules and nanoparticles inside cells. Despite their recognized potential transporting properties, their mode of action is far from being understood and has been a matter of debate. Penetratin, a widely used CPP is one of the first discovered CPPs, yet its mechanism of action remains obscure. Herein an overview on studies regarding cellular and liposomal uptake and the interaction with lipid model systems of CPPs and more particularly penetratin is provided. Special emphasis will be given to biophysical approaches to investigate penetratin/lipid interaction and subsequent lipid reorganization using lipid model systems.