The introduction of microplate-based assays that measure extracellular fluxes in intact, living cells has revolutionized the field of cellular bioenergetics. Here, we describe a method for real time assessment of mitochondrial oxygen consumption rates in primary mouse cortical neurons and astrocytes. This method requires the Extracellular Flux Analyzer Instrument (XF24, Seahorse Biosciences), which uses fluorescent oxygen sensors in a microplate assay format.