Cancer cells exhibit characteristic patterns of metabolic behaviour that can be exploited for therapeutic purposes. Conditions found within the tumour microenvironment, such as hypoxia and selective nutrient availability, are known to influence the metabolism of cancer and stromal cells. Understanding cancer metabolism requires the use of analytical methods that allow detection and quantification of many metabolites simultaneously. Gas chromatography-mass spectrometry (GC-MS) is a versatile method to quantify metabolite abundance and, in combination with stable isotope labelled compounds, can yield important insights into the activity of metabolic pathways in cancer cells. This chapter provides an overview of the use of GC-MS for metabolic analysis of adherent cancer cells with an emphasis on the technical background that should be taken into consideration when designing and executing GC-MS-based metabolomics experiments.