Cells release extracellular vesicles (EVs) into their environment for protection, waste removal, and intercellular communication. As a result, body fluids contain EVs, which are particles with a phospholipid membrane that range in diameter from 30 to 1,000 nm. The molecular composition of an EV depends on the origin and status (e.g. resting, activated, apoptotic) of the releasing cell. The concentration and composition of EVs in body fluids therefore changes with disease, making EVs potential biomarkers. To use EVs as biomarker, there is a need for a technique suitable for clinical application that can 1) detect particles as small as EVs, 2) distinguish EVs from non-EV particles, 3) identify the cellular origin of single EVs, and 4) measure the concentration of EVs in a way that allows comparison between instruments and institutes. Flow cytometry has a high potential to fulfil these requirements. The aim of this thesis is to improve the detection and characterization of single EVs by flow cytometry, to eventually allow the use of EVs as biomarkers in a clinical setting. To that end, this thesis includes methods to 1) lower the scatter detection limit for EVs of a commercial flow cytometer, 2) distinguish EVs from non-EV particles using their refractive index, and 3) compare data from different flow cytometers by applying a calibration method. Scatter detection in flow cytometry is thereby ready to study the biomarker potential of EVs in a clinical setting.