In prion diseases, an abnormal isoform of prion protein (PrP) accumulates in neurons, astrocytes, and microglia in the brains of animals affected by prions. Detailed analyses of PrP-positive neurons and glial cells are required to clarify their pathophysiological roles in the disease. Here, we report a novel method for the detection of PrP in neurons and glial cells from the brains of prion-infected mice by flow cytometry using PrP-specific staining with monoclonal antibody (MAb) 132. The combination of PrP staining and immunolabeling of neural cell markers clearly distinguished neurons, astrocytes, and microglia that were positive for PrP from those that were PrP negative. The flow cytometric analysis of PrP revealed the appearance of PrP-positive neurons, astrocytes, and microglia at 60 days after intracerebral prion inoculation, suggesting the presence of PrP in the glial cells, as well as in neurons, from an early stage of infection. Moreover, the kinetic analysis of PrP revealed a continuous increase in the proportion of PrP-positive cells for all cell types with disease progression. Finally, we applied this method to isolate neurons, astrocytes, and microglia positive for PrP from a prion-infected mouse brain by florescence-activated cell sorting. The method described here enables comprehensive analyses specific to PrP-positive neurons, astrocytes, and microglia that will contribute to the understanding of the pathophysiological roles of neurons and glial cells in PrP-associated pathogenesis. Although formation of PrP in neurons is associated closely with neurodegeneration in prion diseases, the mechanism of neurodegeneration is not understood completely. On the other hand, recent studies proposed the important roles of glial cells in PrP-associated pathogenesis, such as the intracerebral spread of PrP and clearance of PrP from the brain. Despite the great need for detailed analyses of PrP-positive neurons and glial cells, methods available for cell type-specific analysis of PrP have been limited thus far to microscopic observations. Here, we have established a novel high-throughput method for flow cytometric detection of PrP in cells with more accurate quantitative performance. By applying this method, we succeeded in isolating PrP-positive cells from the prion-infected mouse brains via fluorescence-activated cell sorting. This allows us to perform further detailed analysis specific to PrP-positive neurons and glial cells for the clarification of pathological changes in neurons and pathophysiological roles of glial cells.