The present paper describes a colorimetric method of determining the free and total unsaturated fatty acids in animal tissues. The procedure is based in three steps on the following principles : First step is that the tissue homogenates are extracted in chilled acetone in order to eliminate the interfering substance, such as phospholipids, sulfatides and proteins. At next step, after centrifugation acetone layer is decanted and evaporated to dryness. Then the extract is shaken with heptane to solve in the solvent. That the characteristic nature of copper salts of unsaturated fatty acids are freely soluble in heptane and those of saturated acids are not is the bases of separating one from another. Thus unsaturated fatty acids can be isolated in heptane as their salts from saturated acids and other lipid mixture by shaking with copper reagent. Finally the yellowish brown color developed by adding color reagent (0.2% sodium diethyldithiocarbamate in n-butanol solution) which reacts with the copper salts of the acids and produces the color is measured colorimetrically.