Long chain-free fatty acids (LCFFAs) play pivotal roles in various physiological functions, like inflammation, insulin resistance, hypertension, immune cell behavior and other biological activities. However, the detection is obstructed by the low contents, structural diversity, high structural similarity, and matrix interference. Herein, a fast cholamine-derivatization, within 1 min at room temperature, coupled with liquid chromatography-mass spectrometry (LC-MS) approach was developed to determine LCFFAs in complex samples. After derivatization, the ionization and separation efficiency were significantly improved, which resulted in up to 2000-fold increase of sensitivity compared with non-derivatization method, and the limits of detection were at low femtogram level. As well, this approach was applied successfully in the rapid profiling or quantification of targeted and non-targeted LCFFAs in the sera of healthy human and asthma patients. The targeted metabolomics method showed that the contents of 17 PUFAs were significantly changed in asthma patients, especially hydroxyeicosatetraenoic acids (HETEs), hydroperoxyeicosatetraenoic acid (HPETEs) and prostaglandins (PGs). The non-targeted method resulted in the tentatively identification of 35 LCFFAs including 31 saturated and mono-unsaturated LCFFAs, and 4 bile acids, except for 27 poly-unsaturated fatty acids (PUFAs), and the multivariate analysis indicated that eicosapentaenoic acid (EPA), ursodeoxycholic acid, deoxycholic acid, isodeoxycholic acid, palmitic acid, 2-lauroleic acid and lauric acid also have significant difference between healthy and asthma groups except for 17 PUFAs. To the best of our knowledge, this is the first report on the relationship of asthma with 5(S)-, 15(S)-HPETE, 8(S)-, 11(S)-HETE, 15(S)-HEPE, PGA2, PGB2, PGE1, PGF1α, PGJ2, and 13, 14-dehydro-15-keto PGF2α (DK-PGF2α). Copyright © 2017 Elsevier B.V. All rights reserved.