To determine the separation condition for deoxyribonucleosides in RP-HPLC(Reversed-Phase High Performance Liquid Chromatography), the optimization method used was from the relationship of capacity factor and mobile phase composition. In gradient-elution mode by changing mobile phase compositions, the composition and gradient time were determined in stepwise and linear-gradient mode from the relationship. In this work, the binary systems of mobile phase(water/methanol, water/acetonitrile) were utilized. The elution profiles were calculated by the plate theory based on the three retention mechanism of capacity factor. The calculated results showed that in the optimum separation conditions, the mobile phase of water/acetonitrile was maintained for 5 min as 93/7(v/v), then the composition was linearly changed to 91/9 for 5 min.