This chapter will detail methods for the identification and quantification of carotenoid and their esters in food matrices. Carotenoids are the main lipophilic pigments that provide a characteristic orange, red, or yellow color to many fresh fruits and vegetables. They interact positively with human metabolism due to their antioxidant capacity and provitamin A function. The chapter covers the extraction methods, emphasizing using dim or green light to prevent degradation. This is followed by describing the application of high-performance liquid chromatography (HPLC) coupled with diode-array detection and mass spectrometry (MS) that allow an effective separation and identification of carotenoids. This chapter also provides a detailed overview of the various analytical and data interpretation approaches and the considerations and caveats associated with carotenoid structural diversity, choice of authentic standards, interpretation of UV/Vis spectral data, and mass fragmentation patterns for accurate characterization.