Updated project metadata. Exosomes, derived from multivesicular bodies (MVBs), contain proteins and genetic materials from their cell of origin and are secreted from various cells types, including kidney epithelial cells. In general, it is thought that protein cargo is ubiquitylated, but that ubiquitin is cleaved by specific deubiquitylases during the process of cargo incorporation into MVBs. Here, we provide direct evidence that in vivo, deubiquitylation is not essential. Ubiquitin was detected within human MVBs and urinary exosomes by electron microscopy. Protein mass spectrometry identified >6000 proteins in human urinary exosomes, 15% of which were ubiquitylated with various topologies (K63>K48> K11>K6>K29>K33>K27). Ubiquitylated proteins involved in transcriptional regulation or solute transport were significantly enriched. Non-biased analysis of over-represented motifs uncovered a significant preference for basic amino acids upstream of the ubiquitylation site. The current studies demonstrate that in human epithelial cells, deubiquitylation of protein cargo is not necessary for MVB formation and exosome secretion and highlight that urinary exosomes are an enriched source for studying ubiquitin modifications in physiological or disease states.