Targeted metabolomic analysis of fatty acids has linked the dysregulation of fatty acids to many diseases. This study selected five frequently used fatty acid derivatization methods for comparison.We compared the method precisions and derivatization efficiencies, the most economical and best performing method was subjected to method validation. Twenty-four fatty acid standards were used to validate the method, which was later applied to the investigation of potential fatty acid markers of breast cancer.The acetyl chloride method was demonstrated to provide the best derivatization efficiency and lowest cost for plasma samples. The ionic liquid column successfully separated positional and geometric fatty acid isomers within 26min under the optimized conditions. Intra-day and inter-day CVs for most of the fatty acids were <10%. Over 90% of the results showed recoveries within 85%–115%. The validated method was applied to investigate potential fatty acid markers of breast cancer. The fatty acid profiling results revealed that 3 fatty acids (C22:0, C24:0, C18:2n6) were significantly lower in both pre- and post-menopausal breast cancer patients (P<0.05).We demonstrated that the proposed method is an accurate, efficient and economical method for plasma metabolomic studies of fatty acids.