Ayers' method for colorimetric determination of fatty acids was examined and a fundamentally new method was established. The new method is based on the fact that the copper salt of fatty acid dissolves in chloroform in the presence of triethanolamine to show a stable blue color. This can be used for simple and rapid determination of saturated and unsaturated fatty acids of C₁₀-C₂₂ series as a chloroform solution. Ten cc. of 0.0005-0.02M chloroform solution of fatty acid is shaken with 5 cc. of copper-triethanolamine reagent (9 vol. of 1M triethanolamine, 1 vol. of 1N AcOH, and 10 vol. of 5% Cu(NO₃)₂) and optical density of the blue chloroform solution so obtained is measured. This color follows the Lambert-Beer's law within the above concentration range and reproducibility is good. This method can also be applied to the determination of free and conjugated fatty acids in fatty oils and nonionic surface-active agents.