The separation and determination of the major carboxylic acids in wine by liquid chromatography were optimized by using the modelling surface response method. The chromatographic response function was used as a criterion to obtain an optimum peak separation with the minimum analysis time accepted. The starting mobile phase composition (% methanol) and the change in the solvent strength of the mobile phase during the analysis time were considered as experimental parameters. Malic, tartaric, acetic, lactic, succinic and citric acid were determined by linear gradient elution of their phenacyl bromide derivatives. The optimized method was applied to a red wine sample.