Updated publication reference for PubMed record(s): 31337761. Recent years have seen a tremendous increase in the study of extracellular vesicles (EV) geared towards biological understanding, diagnostics and therapy. The interpretation of EV data however remains challenging owing to the complexity of biofluids and the technical variability that are introduced during sample preparation. To understand and mitigate these errors we present the use of traceable recombinant EV (rEV) that mimic endogenous EV both physically and biochemically. The informed use of rEV ensures standardized measurements in various research and biomedical applications. In this dataset we performed mass spectrometry-based proteomics analyses to compare the protein content of endogenous EVs (eEV) and traceable recombinant EV (rEV). The analysis showed that both types of EVs are very similar and that more than 90% of proteins are equally abundant in rEV and eEV isolated from HEK293T cells.