The dissociation constant values of a series of peptides in 5.54, 10, 16.30, 25.03 and 50% (w/w) acetonitrile-water mixed solvents at 298.15 K were determined according to the criteria endorsed by IUPAC. A pronounced change in the acid-base pK values of carboxylic, phenol and thiol groups was observed as the solvent was enriched in acetonitrile. By contrast, pK values of protonated amino-terminal groups were influenced slightly as the solvent was enriched in acetonitrile, although continually increasing pK values were observed. The variation of the pK values obtained, over the whole composition ranged studies, was explained by taking into account the preferential solvation of electrolytes in acetonitrile-water mixtures. To obtain pK values in all possible binary solvent acetonitrile-water mixtures, relationships between pK values and different bulk properties were examined and the Linear Solvation Energy Relationships methodology was applied. The pKa values were then correlated with the Kamlet-Taft, phi, alpha and beta solvatochromic parameters of acetonitrile-water mixtures. The equations obtained allowed calculation of the pK values of peptides in acetonitrile-water mixtures up to 50% (w/w) and thus permitted the acid-base behavior of these substances in the widely used acetonitrile-water media to be known.