Gram-negative and Gram-positive bacteria release a variety of membrane vesicles 
through different formation routes. Knowledge of the structure, molecular cargo 
and function of bacterial extracellular vesicles (BEVs) is primarily obtained 
from bacteria cultured in laboratory conditions. BEVs in human body fluids have 
been less thoroughly investigated most probably due to the methodological 
challenges in separating BEVs from their matrix and host-derived eukaryotic 
extracellular vesicles (EEVs) such as exosomes and microvesicles. Here, we 
present a step-by-step procedure to separate and characterize BEVs from human 
body fluids. BEVs are separated through the orthogonal implementation of 
ultrafiltration, size-exclusion chromatography (SEC) and density-gradient 
centrifugation. Size separates BEVs from bacteria, flagella and cell debris in 
stool; and blood cells, high density lipoproteins (HDLs) and soluble proteins in 
blood. Density separates BEVs from fibers, protein aggregates and EEVs in stool; 
and low-density lipoproteins (LDLs), very-low-density lipoproteins (VLDLs), 
chylomicrons, protein aggregates and EEVs in blood. The procedure is label free, 
maintains the integrity of BEVs and ensures reproducibility through the use of 
automated liquid handlers. Post-separation BEVs are characterized using 
orthogonal biochemical endotoxin and Toll-like receptor-based reporter assays in 
combination with proteomics, electron microscopy and nanoparticle tracking 
analysis (NTA) to evaluate BEV quality, abundance, structure and molecular 
cargo. Separation and characterization of BEVs from body fluids can be done 
within 72 h, is compatible with EEV analysis and can be readily adopted by 
researchers experienced in basic molecular biology and extracellular vesicle 
analysis. We anticipate that this protocol will expand our knowledge on the 
biological heterogeneity, molecular cargo and function of BEVs in human body 
fluids and steer the development of laboratory research tools and clinical 
diagnostic kits.