Ion exchange-chromatography method with the use of hydroxy apatite is considered one of the methods used for the casein fractionation particularly those containing phosphate in its composition. Simplicity of hydroxyapatite-preparation and its easily use in casein fractionation in batches are advantages of this method. Sephadex G-25 or Agarose is used to easement the sample passage through the column. By using of hydroxy-apatite in batches, alpha s-casein can easily separated with the use of buffer solution 0.006 M phosphate at pH 6.6 and the elution is carried out by buffer solution containing 0.25 M KH2PO4, 0.2 M KCl, 4.5 M urea and 0.002 M di-thiotreitol. Before the elution of alpha s-casein, beta- and kappa-casein are separated using buffer solutions containing 0.005 M and 0.1 M KH2PO4 in addition to the previously mentioned components.