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 EV (rEV) as a biological reference material. rEV have similar biochemical and biophysical characteristics to sample EV and function as an internal quantitative and qualitative control throughout analysis. Spiking rEV in bodily fluids prior to EV analysis maps technical variability of EV applications and promotes intra- and inter-laboratory studies. This protocol, which is an Extension to our previously published protocol (Tulkens et al., 2020), describes the production, separation and quality assurance of rEV, their dilution and addition to bodily fluids, and the detection steps based on complementary fluorescence, nucleic acid and protein measurements. We demonstrate the use of rEV for method development, 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.