Carotenoids are a class of fat-soluble compounds that possess an important role in pigmentation of fruits and vegetables, antioxidant activity of food, and important contributors to human health. Their complex chemical structures, cis/trans isomerization, together with ester formation with fatty acids, present a great challenge for extraction and quantification of carotenoids in plant matrices. The limited availability of standardized reference materials, and variation in extraction and quantification methodology also hinders the development of a standard method for accurate quantification of carotenoids. This chapter provides an overview of current methodologies used for carotenoid analysis, including solvent extraction, supercritical fluid extraction, solid-phase extraction, and enzyme-assisted extraction. The advantages and limitations of each method are discussed and highlight the need for standardized reference materials to ensure the reliability of analytical measurements. Additionally, the chapter presents the current challenges and future opportunities in the development of new extraction and quantification methods for carotenoids.