α-Linolenic acid (ALA) and its most important positional isomer γ-linolenic acid (GLA), are essential fatty acids (vitamin F). Therefore, ALA- and GLA-rich edible oils hold great potential in human and animal nutrition, as well as in nutraceutics and cosmetics. Quality control and nutritional validation of oil products is thus of increasing importance. In the present study, the cellulose tris(3,5-dichlorophenylcarbamate)-based chiral stationary phase was successfully used for separation of ALA and GLA, a major challenge in the liquid chromatography of these isomers. The chromatographic conditions were firstly optimized on a HPLC system with UV detection, and the use of a reversed-phase eluent system made up of aqueous 10 mM ammonium acetate/acetonitrile (40/60, v/v; pH6.0) with a 25 °C column temperature resulted optimal for the simultaneous discrimination of the two isomers at a 0.5 mL/min flow rate (α = 1.10; R = 1.21). The method was then optimized for LC-MS/MS implementation. The proposed innovative separation method holds a great potential for the quantification of ALA and GLA in food and biological matrices, thus opening the way to further investigations involving the two positional isomers.