Omega-3 (ω-3) fatty acids are derived from marine sources and offer disadvantages like heavy metal toxicity, unfit for vegetarians, sinking sources of fish, fishy smell, and side effects. Long-term fish oil consumption increases oxidative stress, inflammation, and risk of chronic diseases. Polyunsaturated fatty acids (PUFAs) are abundantly available in plant seeds like Perilla frutescence, Linum usitatissimum, etc. Plant-based PUFAs are either not available in the market or available as complete extracts.The present study was undertaken to standardize the method for extraction and isolation of α-linolenic acid (C18:3, ω-3) from Linum usitatissimum. The gas chromatograph-FID was used to characterise lipids. A complete mixture of lipids was extracted through cold maceration and saponified to separate saturated lipids. The unsaturated lipids, thus obtained were subjected to urea crystallization. Following the segregation of monounsaturated fatty acids, the lipids were esterified by a chemical method to segregate ω-3 PUFAs. The ω-3 PUFAs were subjected to column chromatography to isolate α-linolenic acid (C18:3, ω-3).