After blood, urine is the second most studied biofluid, because it is easy to collect and available in large quantities. However, urine is also the most dynamic biofluid with large variations in concentration of its diverse content. Separation, storage, and characterization of urine extracellular vesicles have special considerations to tackle these large variations of contents by addressing preanalytical (e.g., standardized collection) and analytical factors (e.g., normalization strategies). This chapter will provide real-world, step-by-step protocols for urine collection and storage, along with urine extracellular vesicle cargo isolation and characterization. Single urine extracellular vesicle characterization methods include nanoparticle-tracking analysis (NTA) and flow cytometry. Bulk urine extracellular vesicle characterization methods include protein analysis using immunoblotting and mass spectrometry, and, ribonucleic acid (RNA) analysis with untargeted (e.g., next-generation sequencing) and targeted (e.g., quantitative real-time polymerase chain reaction [PCR]) approaches. This chapter will also discuss urine-specific strategies to address variations in urine concentration, ultralow cargo inputs for downstream analysis, and uromodulin contamination.