The separation condition for five deoxyribonucleosides(dCyd, dUrd, dGuo, dThd, and dAdo) was determined by the optimization developed in this work with RP-HPLC(Reversed-Phase High Performance Liquid Chromatography). The method utilized the newly defined parameter, V_m,1 and the relationship of retention factor and mobile phase composition. In a gradient-elution mode by changing mobile phase compositions, the composition and gradient time were determined in the stepwise-gradient mode. In this work, the binary systems of mobile phase(water/methanol) were applied. The elution profiles were calculated by the plate theory based on the quadratic equation of retention factor, lnk’=AF²+BF+C. F denotes the volume fraction of organic modifier in the mobile phase, while A, B, and C the empirical constants. The optimal mobile phase was obtained by comparing the calculated results of the resolutions of the five components and the retention times of last-eluting component(dAdo). The final result showed that the initial mobile phase was pure water, then after 7 min the second composition of water/methanol, 81/19(v/v,%), was step-changed. In this case, the agreement between the experimental data and the calculated values was good.