The present invention relates to a method for determining the aureomycin premix content and related substances through a high-performance liquid chromatography method, wherein the method adopts the following chromatography conditions that the chromatographic column is (C18, 5 [mu]m) chromatographic column, a mixed solution of water, phosphoric acid and N,N-dimethylformamide according to a volume ratio of 63-77:1.8-2.2:25-30 is adopted as a mobile phase, the phosphoric acid concentration is 2 mol/L, the flow rate is 0.8-1.2 ml/min, the detection wavelength is 270-400 nm, the column temperature is 30-40 DEG C, the sample injection volume is 10-25 [mu]L, and the detector is an ultraviolet detector. According to the method, an aureomycin standard substance solution, a tetracycline standard substance solution, a chlorquatrimycin standard substance solution and an aureomycin premix sample solution are prepared, high-performance liquid chromatography analysis is performed according to the chromatography conditions, and the content of the related substances in the premix is calculated through the peak area. The detection method of the present invention has characteristics of relatively mild chromatography conditions (the mobile phase does not contain the easily-crystallizing salt and does not use the strong acid), easy equipment maintenance, and high test accuracy.