To shorten the time between brain harvesting and microglia isolation, and characterization, we utilized the MACS^® neural dissociation kit followed by OctoMACS^® CD11b magnetic bead isolation technique to positively select for brain microglia expressing the pan-microglial marker CD11b, a key subunit of the membrane attack complex (MAC). This protocol yields a viable and highly pure (>95%) microglial population of approximately 500,000 cells per pup that is amenable for in vitro characterization within hours or days after being harvested from brain tissue. Primary microglia from C57Bl/6 mice were plated for next-day analyses of morphology and cellular markers by immunocytochemistry or for analysis of gene expression under resting or LPS-stimulated conditions. The ease of isolation enables investigators to perform molecular and cellular analyses without having to wait 1–2 weeks to isolate microglia by conventional methods involving mechanical agitation to dislodge these from astrocyte beds.