Knowledge of the protonation constants of dipeptides is important and necessary for complete understanding of their physiochemical behaviour. In this study, the protonation constants of glycylglycine were studied in propylene glycol and dioxan-water mixtures (0 - 60% v/v) at 303.0 ± 0.1 K at an ionic strength of 0.16 M using pH-metric technique. The protonation constants were calculated with the computer program MINIQUAD75 and selection of the best fit chemical models is based on the statistical parameters. The effect of solvent on protonation constants was discussed based on electrostatic forces operating on the protonation equilibria. Distribution of species, protonation equilibria and effect of influential parameters on the protonation constants have also been presented.