The second-stage dissociation constant values of N-(2-acetamido)imino diacetic acid (H2ADA) were determined at 25 ± 0.1 °C by potentiometric titration in different mixed-solvent mixtures (methanol, ethanol, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetone, and dioxane). It was observed that the pKa value is slightly influenced as the solvent is enriched in methanol and ethanol, and remains practically constant in the presence of different amounts of DMF and DMSO. A prounounced change in the pKa value was observed as the solvent is enriched in acetone or dioxane. These results are discussed in terms of various solvent characteristics. It is concluded that the electrostatic effect has only a relatively small influence on the dissociation equilibrium of the imino group [Formula: see text]. Other solvent effects such as solvent basicity, different stabilization of the free base of the conjugate acid by hydrogen bonding interactions in aquoorganic solvent media relative to pure aqueous media, as well as proton–solvent interaction play an important role in the acid dissociation equilibrium.