In the present paper a new methodology has been developed for determination of fatty acids in biological systems using FT-IR spectroscopy. For this method is not necessary chromophore reagent or pre sample preparation. Palmitic acid was chosen as standard, because it is found in several biological systems. The FT-IR spectrum of palmitic acid showed two absorption bands in the region of 2852 and 2920 cm -1 attributed to CH stretching. The results for these bands showed that the Beer-Lambert Law was followed in wide range of concentration of palmitic acid (14 to 257 mmol L -1 ). Potassium ferricyanide (K 3 [Fe(CN) 6 ]) was used as internal standard. Several interferents were tested and only cholesterol, ferric chloride (higher concentration), mixture of amino acids for the band at 2919 cm -1 (higher concentration) and triglyceride could be interferent if they appear in high concentration. Thus, this new methodology has advantage to be not expensive and simple.