Prion diseases or transmissible spongiform encephalopathies are disorders of the central nervous system that affect both humans and animals. The underlying cause of prion diseases is the formation and propagation of the infectious prion protein. Prion diseases are difficult to diagnose and treat due to a prolonged asymptomatic incubation period prior to the onset of clinical symptoms. MicroRNAs (miRNAs) are small noncoding RNA species and have been identified as potential biomarkers that also function to regulate disease-specific pathways and proteins in several neurodegenerative disorders, including prion diseases. Here we describe the quantitative analysis of miRNA isolated from neuronal cells infected with a strain of mouse-adapted human prions. These methods can also be adapted to the discovery of miRNA biomarkers in extracellular vesicles, tissue, and noninvasive biological fluids.