Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic debilitating neuroimmune disease affecting many organs and systems which, in the absence of validated biomarkers, remains diagnosed by clinical criteria. Extracellular vesicles (EV) in blood come from practically all cells in our body and therefore may carry the disease-specific biomarkers needed for the diagnosis of ME. This chapter presents the methodology used on a single pilot study performed to evaluate this possibility to describe a workflow for EV isolation and the analysis of the miRNAs within, which could serve to interrogate additional cohorts of ME/CFS. Among the diverse nature of EV contents miRNAs may constitute a prominent regulatory layer in the development and progress of complex diseases such as ME/CFS, and therefore their study should be further pursued.