AbstractAstrocytes are a heterogeneous population of central nervous system glial cells that respond to pathological insults and injury by undergoing a transformation called “reactivity.” Reactive astrocytes exhibit distinct and context‐dependent cellular, molecular, and functional state changes that can either support or disturb tissue homeostasis. We recently identified a reactive astrocyte sub‐state defined by interferon‐responsive genes like Igtp, Ifit3, Mx1, and others, called interferon‐responsive reactive astrocytes (IRRAs). To further this transcriptomic definition of IRRAs, we wanted to define the proteomic changes that occur in this reactive sub‐state. We induced IRRAs in immunopanned rodent astrocytes and human iPSC‐differentiated astrocytes using TNF, IL1α, C1Q, and IFNβ and characterized their proteomic profile (both cellular and secreted) using unbiased quantitative proteomics. We identified 2335 unique cellular proteins, including IFIT2/3, IFITM3, OASL1/2, MX1/2/3, and STAT1. We also report that rodent and human IRRAs secrete PAI1, a serine protease inhibitor which may influence reactive states and functions of nearby cells. Finally, we evaluated how IRRAs are distinct from neurotoxic reactive astrocytes (NRAs). While NRAs are described by expression of the complement protein C3, it was not upregulated in IRRAs. Instead, we found ~90 proteins unique to IRRAs not identified in NRAs, including OAS1A, IFIT3, and MX1. Interferon signaling in astrocytes is critical for the antiviral immune response and for regulating synaptic plasticity and glutamate transport mechanisms. How IRRAs contribute to these functions is unknown. This study provides the basis for future experiments to define the functional roles of IRRAs in the context of neurodegenerative disorders.