A general equation that relates electrophoretic mobility of polyprotic peptide substances and pH of the running electrolytes is established, taking into account the species in solution and the activity coefficients. Modelling electrophoretic mobility as a function of pH can be simultaneously used for determination of ionization constants and selection of the optimum pH for separation of mixtures of the modelled compounds. The proposed relationships allow an important reduction of the experimental data needed for development of new separation methods. The accuracy of the proposed equations is verified by modelling the migration behavior of a heterogeneous series of polyprotic amphoteric peptide hormones. By calculating the values of predicted resolutions, selection of the optimum pH to perform separation of their mixtures becomes a rapid and simple process.