Twenty years ago, the discovery of peptides able to cross cellular membranes launched a novel field in molecular delivery based on these non-invasive vectors, most commonly called cell-penetrating peptides (CPPs) or protein transduction domains (PTDs). These peptides were shown to efficiently transport various biologically active molecules inside living cells, and thus are considered promising devices for medical and biotechnological developments. Moreover, CPPs emerged as potential tools to study the prime mechanisms of cellular entry across the plasma membrane. This review is dedicated to CPP fundamentals, with an emphasis on the molecular requirements and mechanism of their entry into eukaryotic cells.