Exosomes are small extracellular vesicles released by prokaryotic and eukaryotic cells with a crucial role in cell-to-cell communication in both physiological and pathological conditions. Exosomes contain and transfer active biomolecules, including nucleic acids, proteins and lipids to target recipient cells. In the last decade, many methodologies have been developed for isolating specific exosomal components. In this chapter, we will detail methods to isolate exosomal DNA, considering the crucial role of exosomal DNA in regulating the behavior of recipient cells in multiple settings, including the response of malignant cells to chemo-, radio- and immunotherapy.