Primary glial cell cultures are the most commonly used in vitro model for neurobiological studies. However, the lack of an easy and fast protocol for the isolation and culture of these cells leads to long incubating times with the use of high number of rat brains. Here, we describe a quick protocol based on mixed glial culture for the highly-enriched isolation of microglia, astrocyte and oligodendrocyte cells. The procedure is suitable for beginners as it makes available the easier way to obtain the nervous tissue, the evolution of the mixed culture throughout the time, and the final results after the isolation of each cell type. More than 20×10e6 cells per rat brain can be obtained using this protocol, with no more than 11 days of incubation. This facilitates the use of the primary culture as a tool for researchers to study the development, properties and functions of glial cells in vitro.