Abstract: Objective: Fatty acid benzhydrazide was produced which was used as a reference substance for determination of derivatization yield.The best conditions for the pre- column derivatization of fatty acids using high performance liquid chromatography were studied.Methods: Myristic acid( MA) was selected to represent all of fatty acids( FAs) in the experiment.MA reacted with 2- nitrophenylhydrazine hydrochloride( 2- NPH·HCl) in the case of1- ethyl-3-( 3- dimethylaminopropyl) carbodiimide hydrochloride( 1- EDC·HCl) as coupling agent and pyridine as catalyst,and Myristic benzhydrazide( MBH) was produced. The MBH was separated and purified by silica gel column chromatography and recrystallization.Its structure was identified by MS and1 HNMR.Its purity was detected by TLC and HPLC. Derivatization yield as a indicator,the pre- column derivatization conditions of MA using high performance liquid chromatography were studied.Results: MBH was obtained and it purity was 99.81% which could be used as a reference substance for determination of derivatization yield in the study of optimization of derivatization condition of fatty acid. The best conditions for the pre- column derivatization were as follows: the concentration of 2- NPH·HCl was 0.04 mol / L,the concentration of 1- EDC·HCl was 0.45 mol / L,the temperature of the derivatization reaction was 60 ℃,the time of the derivatization reaction was 15 min.The derivatization yield was99.85% under above the derivatization condition. Linoleic acid derivative was prepared using same derivatization condition.The difference between the peak area of LA derivative and MA derivative was only 1.3%.Conclusion: The optimal pre- column derivatization condition of fatty acid by HPLC was obtained and various fatty acids could be maximum derivatized under above the derivatization condition.