Despite the widespread use of imidazolium-based ionic liquids (ILs) in biotechnology, pharmaceuticals, and green chemistry, their detailed interactions with proteins, particularly affecting structural stability, remain poorly understood. This study examines the effects of ILs on ubiquitin, a thermodynamically robust protein with a β-grasp structure. We found that IL-induced destabilization follows a consistent order with previous findings: [BMIM] > [BMPyr] > [EMIM] for cations and [BF] > [MeSO] > [Cl] for anions. Through pH and ionic strength-dependent studies, we observed that hydrophobic interactions predominantly influence the stability of positively charged ubiquitin, with electrostatic interactions playing a secondary role. NMR studies identified residues impacted by [BMIM][BF]; however, site-directed mutagenesis of these residues showed minimal changes in destabilization, suggesting a global effect. This led us to conduct a broader empirical analysis, incorporating solvent-accessible surface area evaluations, which confirmed that hydrophobic residues are the primary drivers of stability alterations in ubiquitin, with charged residues playing a minimal role. Additionally, single-molecule force spectroscopy results indicate that imidazolium ILs lower the unfolding barrier without altering the transition state structure, offering insights into protein folding dynamics. ILs appear to modulate the stability landscape of proteins by energetically and kinetically favoring the unfolded state over the folded state. These insights offer potential strategies for the selective tuning of protein stability, which could be exploited to modulate protein-protein or protein-substrate interactions in various applications of ILs.