Abstract An efficient optimization method was suggested to separate biologically active peptides by RP-HPLC. In this work, the binary mobile phase of water and acetonitrile was used with the buffer of trifluoroacetic acid (TFA). The elution profiles were calculated by the plate theory based on the linear and quadratic equations of retention factor, ln k=A+BF , ln k=A+BF+CF 2 , and F was the vol.% of acetonitrile. We modified the plate theory to calculate elution profile in both isocratic and gradient mode. From the final calculated results, the first mobile phase composition was water in 0.1% TFA/acetonitrile in 0.1% TFA, 81/19 vol.%, then after 7–8 min, the second composition of mobile phase was linearly changed to 79/21 vol.%, and finally after 8 min, it was kept at the isocratic mode. In the experimental conditions, the agreement between the experimental data and the calculated values was relatively good.