A charged organic-inorganic nanofiltration (NF) membrane prototype was used to separate a mixture of nine amino acids (AA) on the basis of differential electrostatic interactions with the membrane because, for a given pH, some of them were positively charged, some were negative, and some were zwitterions. Effect of pH, amino acid concentration (C-r), and added ionic strength ([NaCl]) on the process selectivity was studied. A global statistical study revealed that pH was the dominant parameter regarding fractionation. C-r and [NaCl] had a weaker effect, but the ratio C-r/[NaCl] demonstrated a pronounced effect on system selectivity. Two split-ups of the mixture were obtained at pH 2 and at pH 12, for a 1-g/L total AA concentration and a C-r/[NaCl] ratio of 0.16. Under these conditions, the differences in transmissions between basic and acid AA were higher than 70%. Interpretation of the results according to the Donnan theory allows us to foresee the potentialities of charged nanofiltration membranes for the fractionation of a complex mixture, such as peptidic hydrolysate to streams containing peptides and amino acids having different isoelectric points.