The analysis of fatty acid methyl esters (FAMEs), derived from food, is a very important characterization procedure. Capillary gas chromatography is the traditionally used technique for the analysis of FAMEs in a wide number of research areas. FAME analysis demands high chromatographic resolution, especially to provide evidence of positional and geometrical isomers of unsaturated fatty acids in complex mixtures. To meet these requirements, polar stationary phases such as polyethylene glycols or cyanopropyl silicones are normally used for the separation of complex FAME mixtures since they enable separation according to unsaturation and carbon number. In recent reviews, the esterification methods, injection techniques, and analytical columns, as well as the analysis and identification of fatty acid isomers, have been described in detail (Gutnikov, J Chromatogr B Biomed Sci Appl 671:71–89, 1995; Sepp€anen-Laakso et al., Anal Chim Acta 465:39–62, 2002; Ruiz-Rodriguez et al., J Pharm Biomed Anal 51:305–326, 2010). Nowadays, gas chromatography (GC) analysis of fatty acids (as methyl esters) in oils (fish oils and vegetable oils) and fat (e.g., milk) is a well-established technique (AOCS, Off Method Ce 1e-91:1–4; Int Stand ISO 15304, 2003). For routine fatty acid analysis, a medium polar cyanopropyl DB-225 column provides excellent separation for complex FAME mixtures. This application is described in this chapter.