During our method development, Rose & Oklander (Rose and Oklander, 1965) and Folch (Folch et al. 1957) methods for lipid extraction were evaluated. The per- centage of lipids extracted using single extractions was 69–75% by Rose and Oklander’s method and 63–72% by Folch’s method. However, the lipids extracted by Folch method were contaminated with heme and would stain the TLC plate as well as contaminate GC injector liner wool. Therefore, we adopted the Rose and Oklander method for the purpose of the study. The challenge dur- ing lipid extraction process was the clumping of red blood cells during the addition of solvent which would inhibit interaction of solvent with the membrane matrix. Therefore, isopropanol was added with continuous vor- texing, giving rise to finer precipitate ensuring deep penetration of solvent within the matrix resulting in im- proved extraction efficiency. The percentage recovery for AA and DHA using single extraction was 46–70% and 54–65% respectively. However, this increased to 72– 86% for AA and 68–81% for DHA with two-step extrac- tion. Three-step extraction further improved the per- centage of lipids (95% and 98% for AA and DHA respectively).