Metabolites produced by an organism can be quite extensive, and one analytical technique alone is not capable of their comprehensive detection and identification. The majority of environmental metabolomic studies have implemented proton nuclear magnetic resonance (1 H-NMR) spectroscopy with little attention given to mass spectrometry (MS) techniques. In this chapter, an analytical technique is outlined that incorporates two-dimensional gas chromatography–time-of-flight MS (GC�GC–TOF-MS) for the identification and quantification of polar metabolites.