In this study, relationships between rooting capability and the changes of endogenous IAA and ABA hormone levels during rooting of hardwood cuttings of 5 BB, 41 B, and 420 A grapevine rootstocks were studied by RP-HPLC. The chromatographic method that we developed was used for the separation and determination of these hormones. For this purpose, the mobile phase composition was optimised in order to separate IAA (indole-3-acetic acid) and ABA (abscisic acid). Mobile phase was optimised by establishing relationships between retention parameters and single solvent polarity parameter. pH measurements in acetonitrile-water binary mixtures were carried out by using IUPAC rules. Different levels of IAA and ABA substances correspond to the different rooting ability of 5 BB, 41 B and 420 A hardwood cuttings. Low IAA and high ABA levels were detected in cuttings of 41 B and 420 A having low rooting rate, whereas high IAA and low ABA levels were detected in cuttings of 5 BB, having high rooting rate in dormant stage. In general, endogenous IAA increased, while ABA decreased during rooting process. It was found that there was a positive correlation between IAA level and rooting rate, but a negative correlation between ABA level and rooting rate.