Dimethyl sulfoxide (DMSO) has a significant, multi-faceted role in medicine, pharmacy, and biology as well as in biophysical chemistry and catalysis. Its physical properties and impact on biomolecular structures still attract major scientific interest, especially the interactions of DMSO with biomolecular functional groups. In the present study, we shed light on the “isolated” carboxylic (–COOH) and amide (–NH) interactions in neat DMSO via ¹H NMR studies along with extensive theoretical approaches, i.e. molecular dynamics (MD) simulations, density functional theory (DFT), and ab initio calculations, applied on model compounds (i.e. acetic and benzoic acid, ethyl acetamidocyanoacetate). Both experimental and theoretical results show excellent agreement, thereby permitting the calculation of the association constants between the studied compounds and DMSO molecules. Our coupled MD simulations, DFT and ab initio calculations, and NMR spectroscopy results indicated that complex formation is entropically driven and DMSO molecules undergo multiple strong interactions with the studied molecules, particularly with the –COOH groups. The combined experimental and theoretical techniques unraveled the interactions of DMSO with the most abundant functional groups of peptides (i.e. peptide bonds, side chain and terminal carboxyl groups) in high detail, providing significant insights on the underlying thermodynamics driving these interactions. Moreover, the developed methodology for the analysis of the simulation results could serve as a template for future thermodynamic and kinetic studies of similar systems.