Urinary exosomes are small (<100 nm) vesicles secreted into urine from renal epithelial cells. They are coated with lipid bilayer, they contain an array of membrane and cytosolic proteins, and selected RNA species, reflecting the molecular composition of their cell of origin. Thus, urinary exosomes have received considerable attention as potential biomarker source, as their proteomic analysis could lead to the discovery of new non-invasive site-specific biomarkers for renal diseases. Here, we describe a robust method for urinary exosome preparation, additional protocols for their biochemical characterization and for the quantitation of different preparations, to be used for comparative proteomic studies.