A preparative reversed-phase high performance liquid chromatography-electrospray ionization mass spectrometry (RP-HPLC/ESI-MS) method to obtain low cost casein peptides was developed. Casein was hydrolyzed by trypsin and the hydrolysate was analyzed by RP-HPLC/ESI-MS firstly. The gradient elution program in an analytical column was optimized to achieve a better separation. The effect of pH of the mobile phase on the separation was also investigated. Then, the optimal analytical conditions were applied in a preparative column directly. In each loading of the hydrolysate, several pure peptide fractions were collected automatically by the inductions of ultraviolet absorbance and mass spectrometry signals together. The process is simple and effective. The influence of the loading amount in a preparative column was investigated intensively. For the collection consisting of hydrophilic peptides that were poorly separated in the primary process, secondary separation was performed. By changing the gradient elution, several different pure peptides were obtained.