The protonation equilibria of vitamin B9 (folic acid) was studied at 298.15 K in a different water-dioxane mixtures [100 w(dioxane) = 20 %, 40 %, 60 %, and 80 96] with an ionic strength of 0.15 mol.dm(-3) NaNO3. The influence of dioxane content on the protonation processes was explained. Also, four protonation constants have been determined in 60 % dioxane with an ionic strengths of (0.15, 0.20, 0.25, and 0.30) mol.dm(-3) NaNO3 using the pH-potentiometric technique. HYPERQUAD 2008, a program based on nonlinear least-squares curve fitting was used to determine these four stepwise protonation constants of folic acid from the analysis of pH-potentiometric data. From the determined protonation constants, the representative folate species distribution diagrams were provided by HYSS 2009 program and discussed.