In addition to soluble proteins, cells secrete various types of membrane-enclosed extracellular vesicles (EVs: exosomes, ectosomes and others) and non-vesicular nanoparticles (ENPs) that are thought to play a role in intercellular communication. We exhaustively characterized the protein composition of secreted EVs and ENPs of murine tumor cell lines, including thorough separation of small EVs from co-isolated virus-like particles.