Extracellular vesicles (EVs) are small membrane-bound particles released into extracellular space by almost all cell types, and found in body fluids like blood, urine, and saliva. Mounting evidence has demonstrated the clinical potential of EVs as diagnostic and therapeutic tools to analyse physiological/pathological processes due to their ability to transport biomolecules secreted from diverse tissues of an individual.For example, the urinary EVs (uEVs), released from all regions of the kidney’s nephron and from other cells that line the urinary tract, retain proteomic and transcriptomic markers specific to their cell of origin representing a valuable tool for kidney disease diagnosis.Despite the numerous efforts in developing suitable methods to separate EVs from biofluids, providing material of high purity and low variability poses a limit to clinical translation.This chapter focuses on advantages and disadvantages of several EV isolation methodologies, and provides examples of uEV isolation protocols based on time, cost, and equipment considerations, as well as the sample requirements for any downstream analyses.