A method for the determination of fatty acids in serum based on GC-MS (micro-SIS detection mode) has been developed and the separation and cis/trans isomers have been identified. A prior two-step extraction/derivatization procedure accelerated by ultrasound allows individual determination of esterified (EFAs) and non-esterified fatty acids (NEFAs), and shortening of the derivatization steps to 5 min for EFAs and 15 min for NEFAs. The total analysis time for 39 fatty acids was 61 min. The minimum LOD and LOQ values were 0.002 and 0.006 microg/ml, respectively. The proposed method was validated for EFAs and NEFAs using two different methods and the results show no statistical differences between the proposed method and those used as reference. The proposed derivatization-extraction methodology is suitable for fatty-acid analysis of human serum, and can be applied to nutritional and epidemiological studies.