The isolation and analysis of microRNAs (miRNAs) contained in microvesicles and in particular nano-sized exosomes has become an increasingly important tool to understand their widespread impact in various fundamental and interactive cellular processes. Fundamental studies regarding exosome biogenesis and miRNA sorting may ultimately unravel their potency as a promising class of highly specific disease biomarkers. Here we describe the methods and protocols used in our laboratory to isolate and purify exosomes, how we extract the (small) RNA content, how to analyze copy numbers, and finally how to measure exosome-mediated transfer of these molecules into recipient cells. Our techniques have been optimized for the detection of Epstein–Barr virus (EBV)-encoded miRNAs that are loaded into exosomes. We discuss how a focus on EBV-miRNA detection may yield important new clues into exosome-mediated cross talk by B cells in humans.