Introduction: The capability of cell-penetrating peptides (CPP), also known as protein transduction domains (PTD), to enter into cells possibly with an attached cargo, makes their application as delivery vectors or as direct therapeutics compelling. They are generally biocompatible, nontoxic, and easy to synthesize and modify. Three decades after the discovery of the first CPPs, similar to 2,000 CPP sequences have been identified, and many more predicted. Nevertheless, the field has a strong commitment to authenticate new, more efficient, and specific CPPs. Areas covered: Although a scattering of CPPs have been found by chance, various systematic approaches have been developed and refined over the years to directly aid the identification and depiction of new peptide-based delivery vectors or therapeutics. Here, the authors give an overview of CPPs, and review various approaches of discovering new ones. An emphasis is placed on in silico methods, as these have advanced rapidly in recent years. Expert opinion: Although there are many known CPPs, there is a need to find more efficient and specific CPPs. Several approaches are used to identify such sequences. The success of these approaches depends on the advancement of others and the successful prediction of CPP sequences relies on experimental data.