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Summary: Mitochondrial damage-associated molecular patterns (mitoDAMPs) are released from cells dying uncontrolled, non-apoptotic deaths, usually secondary to disease or trauma. Here, we describe preparation of mitoDAMPs from mouse liver, but this protocol can be adapted for preparation of mitoDAMPs from other species and tissues. Tissues are dissociated and then processed to isolate mitochondria. Mitochondria are then sonicated and mitoDAMPs are collected by ultracentrifugation. This procedure produces μg quantities of mitoDAMPs and facilitates research to understand their impacts in health and disease.For complete details on the use and execution of this protocol, please refer to Westhaver et al. (2022). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.