 Exosomes are small extracellular vesicles secreted by all cell types playing intercellular communication roles in health and disease by transferring cellular cargos such as functional proteins, metabolites and nucleic acids to recipient cells. An in-depth understanding of exosome biology is therefore essential to ensure clinical development of exosome-based investigational therapeutic products. In this review article, we summarise the most up-to-date knowledge about the complex biological journey of exosomes from biogenesis and secretion, transport and uptake to their intracellular signalling. We delineate the major pathways and molecular players that influence each step of exosome physiology, highlighting the roots of interest, which will be of benefit to exosome manipulation and engineering. We highlight the main controversies in the field of exosome research, their adequate definition, characterisation and biogenesis at plasma membrane. We also delineate the most common identified pitfalls affecting exosome research and development. Unravelling exosome physiology is key to their ultimate progression towards clinical applications. This article was authored by Sonin Burung, Danny Perico, Lukia Taramanidou and others. We are article.tv, links in the description below.