Microglia are the innate immune cells of the central nervous system. Although numerous methods have been developed to isolate microglia from the brain, the method of dissociation and isolation can have a profound effect on the function of these highly dynamic cells. Here, we present an optimized protocol to isolate CD11b+ cells (microglia) from mouse or human brain tissue using magnetic bead columns. Isolated microglia can be used to model diseases with neuroinflammatory components for potential therapeutic discoveries. For complete details on the use and execution of this protocol, please refer to Hanamsagar et al. (2017), Rivera et al. (2019), and Edlow et al. (2019).