Updated project metadata. JC polyomavirus (JCPyV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a devastating demyelinating disease of the central nervous system that results in the widespread formation of lesions across the brain parenchyma. The virus is opportunistic and remains in a benign state in the kidneys and lymphoid organs of more than half of the global human adult population. However, in rare cases of severe or selective immune suppression, JCPyV can establish a lytic infection of glial cells in the brain. While PML has traditionally been characterized as a lytic infection of oligodendrocytes, more recent findings suggest an important role for astrocytes during the initial stages of disease. Because of the exceptional species and tissue specificity of the virus, appropriate models of JCPyV infection in the brain are lacking, thus hampering progress towards the development of novel antiviral strategies and biomarkers of disease activity. Towards the aim of biomarker development extracellular vesicles (EVs) were isolated from JCPyV-infected and mock-infected human iPSC-derived astrocytes and analyzed by LC-MS/MS. As a inflammatory control, EVs were also isolated from cytokine-stimulated iPSC-derived astrocytes. We demonstrate that the proteomic signature associated with EVs from JCPyV-infected astrocytes reflect what was observed on a cellular level for infected astrocytes while being strikingly different from that of EVs generated under inflammatory conditions.