Sterols are minor components of food lipids, often used as markers for confirming the authenticity of oils and food, so their correct characterization and quantification are very relevant for detecting adulterations. In addition, plant sterols can be used for the formulation of functional foods due to their cholesterol-lowering properties, so an accurate analytical determination of this compound class is also required for food labeling. Here, we describe the analytical determination of food sterols, including the various preparative steps (extraction, cold saponification, purification) and the gas chromatographic (GC) analysis with different detectors (flame ionization detector (FID) and mass spectrometer (MS)).