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Metabolite profiling is a rapidly expanding technology which aims to quantify the entire metabolome of biological samples. Gas Chromatography Mass Spectrometry (GC-MS) is one of the most widely used analytical tools for profiling highly complex mixtures of primary metabolites, such as organic and amino acids, sugars, sugars alcohols, phosphorylated intermediates, and lipophilic compounds. This chapter summarizes all of the preparatory steps for metabolite profiling of polar compounds by GC-MS in tomato fruit, from the sampling of plant material to the derivatization procedures required to render the metabolites volatile.