Deep eutectic solvents (DESs) have emerged as sustainable alternatives to traditional organic solvents for extraction and separation applications. This chapter explores the fundamental principles and innovative uses of DESs in the selective extraction and separation of pharmaceuticals, bioactive compounds, metals, pesticides, food additives, and other valuable materials from complex matrices. These methods employ the unique physicochemical properties of DESs, including low volatility, tunable polarity, and high solubilization capacity, to develop environmentally friendly techniques for extraction and separation processes. DESs have been successfully utilized as an eluent in solid-phase extraction, as an extractant or disperser in liquid–liquid extraction and microextraction, as a mobile phase modifier in high-performance liquid chromatography, as chiral selectors or auxiliary additives in capillary electrophoresis, and as stationary phases in gas chromatography. The applications of DESs as an eco-friendly solvent and their roles in reducing hazardous waste and enhancing operator safety have been discussed in this chapter. Future research directions are proposed to address current challenges and broaden the applications of DESs in separation technologies.