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Summary: Understanding the nutritional and immunomodulatory components of breast milk is crucial to developing novel mechanisms to optimize neonatal health. Here, we present a protocol to express and isolate murine milk in sufficient quantities for further analysis of components and bioactivity. We describe steps for separating dams from pups, administering intraperitoneal anesthetic and oxytocin, and expressing milk using a minimally modified and readily available commercial breast pump parts.For complete details on the use and execution of this protocol, please refer to Meyer et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.