Abstract: A modified method for determination of free fatty acids (FFAs) in human serum was established by gas chromatography time-of flight mass spectrometry (GC-TOF-MS). Heptadecanoic acid methyl ester was selected as internal standard. After derivatization by 2.5% H^₂SO₄/CH₃OH, the FFAs in serum were transformed into the fatty acid methyl esters, and N-hexane was added to extract the products. The analysis of extract was performed by GC-TOF-MS. Better results were obtained with derivatization comparing with non-derivatization. At least 20 kinds of FFAs (C₁₂-C₂₄) in human serum are determinated by the modified method. The analysis is completed within 33 min. The modified method shows potential useful for the scientific research to the disease correlating FFAs and lipidomics due to the simplify, rapidity, high accuracy and enormous information.