Cells secrete different types of membrane vesicles (MVs), which may act as important entities in normal human physiology and in various pathological processes. The established methods for quantification of MVs require purification or preanalytical handling of samples with labeling moieties. The authors' aim was to develop a method for high-throughput, labeling-free quantification of nonpurified MVs. Scanning ion occlusion sensing technology, which relies on the detection of particles upon their movement through a nanopore, was investigated for the ability to quantify nanosized MVs (<400 nm) in bodily fluids and cell culture supernatants. Scanning ion occlusion sensing allowed for rapid and easy measurement of the concentration of MVs in all biological fluids tested. Scanning ion occlusion sensing technology enables the quantification of MVs in biological samples without the requirement of MV isolation and/or labeling. This offers a highly valuable addition to the currently used repertoire of MV quantification methods.