Many organisms use osmolytes to protect their cells from adverse environmental conditions. Osmolytes help to stabilize the structure of proteins and to maintain their function. We studied the binding of the competitive inhibitor proflavine to α-chymotrypsin in the presence of six biologically relevant osmolytes (TMAO, glycine, sarcosine, N,N-dimethylglycine, betaine, and glycerol). To investigate the role of osmolytes in protein-ligand binding, we estimated the relative residence times of the ligand at the catalytic site of the enzyme, using τ-random acceleration molecular dynamics and carried out extensive molecular dynamics simulations. The computational studies were complemented with UV/Vis, circular dichroism (CD) and fluorescence spectroscopy studies as well as isothermal titration calorimetry (ITC) measurements. We found that the osmolytes can modulate the interactions between proflavine and the catalytic triad of the enzyme by modifying the solvent environment at the active site. The combination of computational and experimental studies allowed identifying two main types of behavior among the osmolytes: the experimental binding constants and computed ligand residence times are higher in the presence of glycine, sarcosine, N,N-dimethylglycine, and glycerol. By contrast, in the presence of TMAO and betaine, the computed ligand residence times and the experimental binding constants of the α-chymotrypsin-proflavine complex are lower. The strong correlation found by us between computed ligand residence times and experimentally determined binding constants in the presence of different osmolytes is particularly relevant since the identification of parameters that directly correlate to substrate binding can provide a guide for solvent selection. Indeed, on this basis, we delivered computational predictions, concerning the binding constant of proflavine to α-chymotrypsin in 0.5 M DMSO, which we experimentally corroborated. Our comprehensive study provides a molecular rationale for the pivotal role that osmolytes play in modulating ligand binding as well as key insights that could aid the design of the liquid media for biocatalytic systems.