Cell‐penetrating peptides (CPPs) encompass a class of peptides that possess the remarkable ability to cross cell membranes and deliver various types of cargoes, including drugs, nucleic acids, and proteins, into cells. For this reason, CPPs are largely investigated in drug delivery applications in the context of many diseases, such as cancer, diabetes, and genetic disorders. While sharing this functionality and some common structural features, such as a high content of positively charged amino acids, CPPs represent an extremely diverse group of elements, which can differentiate under many aspects. In this review, we summarize the most common characteristics of CPPs, introduce their main distinctive features, mechanistic aspects that drive their function, and outline the most widely used techniques for their structural and functional studies. We highlight current gaps and future perspectives in this field, which have the potential to significantly impact the future field of drug delivery and therapeutics.

Small extracellular vesicles (sEVs) present fairly distinctive lipid membrane features in the extracellular environment. These include high curvature, lipid‐packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. sEV membrane could be then considered as a “universal” marker, alternative or complementary to traditional, characteristic, surface‐associated proteins. Here, we introduce the use of membrane‐sensing peptides as new, highly efficient ligands to directly integrate sEV capturing and analysis on a microarray platform. Samples were analysed by label‐free, single‐particle counting and sizing, and by fluorescence co‐localisation immune staining with fluorescent anti‐CD9/anti‐CD63/anti‐CD81 antibodies. Peptides performed as selective yet general sEV baits and showed a binding capacity higher than anti‐tetraspanins antibodies. Insights into surface chemistry for optimal peptide performances are also discussed, as capturing efficiency is strictly bound to probes surface orientation effects. We anticipate that this new class of ligands, also due to the versatility and limited costs of synthetic peptides, may greatly enrich the molecular toolbox for EV analysis.