Updated project metadata. Extracellular vesicles (EV) in body fluids are extensively studied as potential biomarkers for numerous diseases. Major impediments of EV-based biomarker discovery include the manual labor, and the specificity and reproducibility of EV sample preparation. To tackle this, we present an automated liquid handling workstation for the density-based separation of EV from human body fluids and compare its performance to manual handling by (in)experienced researchers. To validate automated density-based separation of EVs from human body fluids, including blood plasma and urine, we assess variation, yield and purity by mass spectrometry-based proteomics.