The experimental procedure involved in metabolic mapping of astrocytes and neurons in culture using stable isotopes and gas chromatography-mass spectrometry (GC-MS) comprises several steps each of which has to be thoroughly considered in order to obtain the desired information. In this chapter we describe the different aspects to be considered when designing an incubation experiment to provide information about cellular metabolism in vitro, i.e., incubation time, incubation medium, and which isotope to employ. The labeling patterns obtained in several metabolites following metabolism of a variety of ¹³C or ¹⁵N labeled precursors through different metabolic pathways are depicted in order to provide suffi cient insight to enable the reader to select the best suited precursor. The cell extraction and sample preparation procedures required before GC-MS analysis are described as are the subsequent integration of chromatograms and the calculations needed to correct for natural abundance and to obtain percentage labeling of M, M + 1, M + 2, etc. in a compound. Also, the cell culturing procedure for preparing primary cultures of neurons and astrocytes and also co-cultures of these cell types isolated from either mouse cerebral cortex or cerebellum is described in detail.