Primary cultures are an important in vitro tool to study cellular processes and interactions. These cultures are complex systems, composed of many cell types, including neurons, astrocytes, oligodendrocytes, microglia, NG2 cells, and endothelial cells. For some studies it is necessary to be able to study a pure culture of one cell type, or eliminate a particular cell type, to better understand its function. There exist cell culture protocols for making pure astrocyte or microglia cultures. Here we present two protocols to produce cultures depleted for microglia: in the first case, from a mixed astrocyte-microglia culture and, in the second, for eliminating microglia from neuronal cultures.