Linoleic acid, an omega-6 fatty acid present widely in plant seeds and one of the major components among the fatty acids in Linum Usitatissimum L., commonly called as flax seed and represents the content of 12–19% out of the total lipids in flax seed. This work attempted to quantify the Linoleic acid from flax seed oil using the high-performance thin layer chromatography (HPTLC) method with mobile phase, methanol: toluene: formic acid (3:7:1 v/v), and the wavelength used is 254 nm. The method gave a sharp, symmetrical peak at Rf value 0.63 ± 0.02. The method was validated using intracerebral brain hemorrhage (ICH) guidelines and applied to the assay of marketed capsules of flaxseed. It is confirmed that the reported method can help analyze flax seed oil in different dosage forms, along with cosmetic products containing linoleic acid-rich oils.