Context <p>Biodiesel has emerged as a sustainable and viable alternative to fossil fuels to meet the growing global energy demand. However, crude biodiesel produces glycerol as a major byproduct, which adversely affects its quality and engine performance. Additionally, the combustion of biodiesel in the presence of glycerol emits harmful pollutants, making the effective removal of glycerol a critical step in biodiesel purification. Liquid–liquid extraction using conventional organic solvents has been explored for glycerol removal from biodiesel, but its applicability is limited by high volatility, flammability, poor selectivity, and losses of biodiesel, which compromise process safety and energy efficiency. Consequently, recent attention has shifted toward sustainable solvents, including deep eutectic solvents, which offer low volatility, reusability, and improved separation performance. In this study, deep eutectic solvents are explored as promising extraction solvents for the selective removal of glycerol from biodiesel.</p> Methods <p>In this study, classical molecular dynamics simulations were performed with the GROMACS package and the OLPS-AA force field to investigate the molecular interactions governing glycerol extraction from biodiesel using deep eutectic solvents. The DESs studied included choline chloride:urea (1:2), choline chloride:ethylene glycol (1:2), and choline chloride:ethylene glycol (1:3). Structural and dynamical properties were analyzed using radial distribution functions, hydrogen-bond analysis, and density profiles to quantify intermolecular interactions and preferential solvation behavior. The molecular-level insights obtained from these simulations were used to assess the affinity of deep eutectic solvents for glycerol by determining the glycerol distribution coefficient between the DES-rich and biodiesel-rich phases.</p>