Microglia are the resident immune cells of the central nervous system, responsible for defending against infections, responding to tissue damage, and maintaining homeostasis. Dysregulation of their phagocytic activity has been implicated in many neurodegenerative diseases of the brain and the eye, including Alzheimer's disease, multiple sclerosis, glaucoma, and age-related macular degeneration. Past work has shown that brain and retinal microglia switch to a disease-associated molecular phenotype (DAM) following phagocytosis of apoptotic neurons in vivo, but it is technically challenging to perform these assessments and isolate phagocytic cells for downstream analysis. Although several protocols exist for isolating and culturing microglia to evaluate their phagocytic activity in vitro, these approaches often fail to mimic the pathophysiology of neurodegeneration, where dysregulated phagocytosis of damaged or apoptotic neurons is a key feature. Here we describe a protocol for evaluating phosphatidylserine-mediated phagocytosis by primary mouse brain microglia in vitro. The procedure involves isolating primary mouse brain microglia, inducing apoptosis in a feeder neuronal cell line, labeling apoptotic cells with a fluorescent dye, feeding the labeled cells to the microglia, and analyzing phagocytosis using several complementary approaches. This protocol enables an accessible and high-throughput assessment of microglial phagocytic activity using a biologically relevant stimulus, offering improved insights into the regulation of this critical immune process in a disease-relevant context.