Carotenoids and tocochromanols are lipid-soluble secondary plant metabolites that are essential for the normal functioning of plants and, in some cases function as a source of vitamin A and E in humans. Enhancement of the provitamin A carotenoid and tocochromanols levels in sorghum and other cereal grains through traditional breeding and transgenic methods has increased in recent years with interest in biofortification of grains to combat micronutrient deficiencies in developing countries. With this increase in research, reliable methodology for the extraction, identification, and quantification of individual carotenoids and tocochromanols species from sorghum and other cereal grains is essential. Here, we describe a basic method for extraction of carotenoid and tocochromanols adapted to sorghum grain and chromatographic condition for separation, identification, and quantification of individual carotenoid using High-Performance Liquid Chromatography (HPLC) system.