Abstract: A method of high performance liquid chromatography for the determination of antioxidants in food in the national standard GB 5009.32-2016 was optimized. The optimized test conditions were as follows: Samples were dissolved in n-hexane saturated with acetonitrile, and the antioxidants were extracted 3 times by 10 mL of acetonitrile saturated with n-hexane (containing Ascorbyl Palmitate); the C₁₈ solid phase extraction column was used for purification. The Agilent Eclipse XDB-C₁₈ column (250 mm×4.6 mm, 5 μm) was used as stationary phase, and 0.1% (volume fraction) formic acid-acetonitrile mixtures in different volume ratios were used as mobile phase for gradient elution. The optimized results showed that the 9 antioxidants were completely separated within 30 min, the peak shapes were symmetrical, and the resolution was better, which solved the problems including the poor peak shape of PG and the failure of the baseline separation of THBP and TBHQ in the national standard method. The spiked recovery increased from 31.4%-90.1% of the national standard method to 62.0%-104%, and RSDs (n=6) of the determined values were all less than 13%.