Exosomes are small membrane encapsulated vesicles released by cells during normal and stress (pathological) conditions that may play multiple biological roles. They contain typical cellular components, including phospholipids, cholesterol, proteins, glycoconjugates, nucleic acids and metabolites. A great deal of interest has risen about the possibility that they are an alternate form of intracellular communication. However, the increasing attraction has been centered on the prospect that exosomes could become disease biomarkers as part of the new concept of liquid biopsies. In this regard, attention has been directed at investigating the content of exosomes within urine, since this is an ideal body fluid because it could be collected in great quantities, recurrently, and with minimal intervention. Although urine exosomes are very abundant, their isolation has been challenging due to the contamination with many soluble factors within the fluid. Several methods have been developed with different degrees of success. In addition, a major effort has been directed at characterizing all components of urine exosomes.