In an effort to reduce the total amount of organic solvent required for the elution of fat soluble vitamins in reversed-phase liquid chromatography separation, a systematic approach was developed using a mixture of methanol, water and alkanol as the mobile phase. In the optimization procedure, a five-parameter retention model was developed and the statistical evaluation of model, revealed that, the variations of retention times of each vitamins caused by addition of alkanols to the mobile phase were predicted with an average relative deviation of 1.83%. For this model, all the R 2 adj and R 2 pred values for all vitamins were higher than 0.9839 and 0.9798, respectively and LOF values were not statistically significant. A mobile phase containing methanol: octanol: water (86:9:5) was proposed as the optimum mobile phase composition using grid search method and a good agreement were observed between predicted and experimental response values (relative error= -0.734%). Using this optimum condition, the simultaneous separation of six fatsoluble vitamins, was achieved in 9.94 min. The results showed an improvement in the separation of fat soluble vitamins and in reduction of the total amount of organic solvent used for separation with addition of low amounts of alkanols as additive to the mobile phase.