Updated project metadata. The diagnostic and therapeutic use of extracellular vesicles (EV) is under intense investigation and may lead to societal benefits. Reference materials are an invaluable resource for developing, improving and assessing the performance of regulated EV applications and for quantitative and objective data interpretation. We have engineered recombinant extracellular vesicles (rEV) as a biological reference material. rEV have similar biochemical and biophysical characteristics as sample EV and function as an internal quantitative and qualitative control throughout analysis. Spike-in applications of rEV in bodily fluids prior to EV analysis map technical variability of EV applications and promote intra and inter laboratory studies. This protocol describes the production, recovery and quality assurance of rEV, their dilution and addition to bodily fluids, and the detection steps based on fluorescence-, nucleic acid- and protein measurements. Multiple application potentials for rEV are exemplified, including method development, big data normalization and assessment of pre-analytical variables. The protocol can be adopted by researchers with standard laboratory and basic EV separation/characterization experience and requires ~4–5 d.