The invention relates to an assay method of the content of fat-soluble vitamins in concentrated feed, which belongs to the technical field of chemical ingredient assay. The method comprises the following steps: tert-butylhydroquinone is added into a concentrated feed sample dissolved in a solvent, and the mixture is ultrasonically treated, filtered and concentrated, so that a to-be-assayed solution is obtained; a standard fat-soluble vitamin product is used for substituting for the concentrated feed sample, and by the same treatment, a standard solution is obtained. High performance liquid chromatography is adopted to respectively assay the to-be-assayed solution and the standard solution, and according to obtained high performance liquid chromatograms, the content of fat-soluble vitaminsin the concentrated feed sample is calculated. The fat-soluble vitamins include vitamin A, vitamin D3 and vitamin E. On one hand, the process of the assay method is simple and easy to operate, the sample preparation time is short, the oxidation of the fat-soluble vitamins in the process of pretreatment is effectively prevented, and because the usage of toxic and harmful ether is abandoned, the assay method is safe and environmentally friendly; and on the other hand, the assay method has high assay precision and a good effect, and realizes the simultaneous separation and assay of the vitamins within a short time.