This protocol describes an analytical platform for the analysis of intra- and extracellular metabolites of microbial cells (yeast, filamentous fungi and bacteria) using gas chromatography-mass spectrometry (GC-MS). The protocol is subdivided into sampling, sample preparation, chemical derivatization of metabolites, GC-MS analysis and data processing and analysis. This protocol uses two robust quenching methods for microbial cultures, the first of which, cold glycerol-saline quenching, causes reduced leakage of intracellular metabolites, thus allowing a more reliable separation of intra- and extracellular metabolites with simultaneous stopping of cell metabolism. The second, fast filtration, is specifically designed for quenching filamentous micro-organisms. These sampling techniques are combined with an easy sample-preparation procedure and a fast chemical derivatization reaction using methyl chloroformate. This reaction takes place at room temperature, in aqueous medium, and is less prone to matrix effect compared with other derivatizations. This protocol takes an average of 10 d to complete and enables the simultaneous analysis of hundreds of metabolites from the central carbon metabolism (amino and nonamino organic acids, phosphorylated organic acids and fatty acid intermediates) using an in-house MS library and a data analysis pipeline consisting of two free software programs (Automated Mass Deconvolution and Identification System (AMDIS) and R).