Co-solvents strongly influence the solute-solvent interactions of biomolecules in aqueous environment and have profound effects on the stability and activity of several proteins and enzymes. Experimental studies have previously reported on the hydrogen-bond dynamics of water molecules in the presence of co-solvent, but understanding the effect from a solute’s perspective could provide a greater insight towards protein stability. Our study uses 2D IR spectroscopy to investigate how DMSO as a co-solvent affects the dynamics of hydrogen-bonding interactions between the C=O group of ethyl acetate and water by quantitatively estimating the hydrogen-bond exchange rate with and without DMSO in aqueous solution. 2D IR results show spectral signatures of a chemical exchange process; the presence of the co-solvent is found to lower the hydrogen-bond exchange rate by a factor of 5. The measured exchange rates are 6.68×1011 s-1 and 1.48×1011 s-1 in neat water and 1:1 DMSO/water, respectively. The widely different hydrogen-bond lifetimes and exchange rates with and without DMSO indicate a significant change in the ultrafast hydrogen-bond dynamics in the presence of a co-solvent, which in turn might play an important role in the stability and activity of biomolecules.