Plant hormones, specialized chemical substances produced by plants, are the main internal factors controlling growth and development. In this study the pH and polarity of the mobile phase were taken into consideration to optimize the mobile phase for the chromatographic separation of 3 important plant hormones: abscisic acid (ABA), indole-3-acetic acid (IAA) and gibberellic acid (GA(3)). pK(a) values of ABA, IAA and GA3 were determined using retention factors. These 3 hormones were extracted from 99 R (Vitis berlandieri x Vitis rupestris) and rose oil (Rosa damascena Mill.) and the chromatographic method developed was used for the separation of these hormones.