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Summary: The immune fraction of the tumor microenvironment has been proven to play a fundamental role in glioblastoma progression and therapeutic response. Here, we present a detailed magnetic-bead-enrichment-based protocol to isolate and analyze the composition of this fraction from mouse brain tumors. The protocol is optimized to achieve high yields of viable immune cells. We also detail characterization of the immune subtypes by FACS analysis. Our procedure is applicable for either lentiviral-induced tumors or transplant models in syngeneic immunocompetent mice.For complete details on the use and execution of this protocol, please refer to Magod et al. (2021).