The invention discloses a method for detecting related substances of polysorbate 80. The method comprises the following steps of: (a) preparation of the solution of a reference substance; (b) the preparation of the solution of a test sample; (c) chromatographic condition and system applicability test; (d) determination method, namely precisely measuring 20 microliters of the solution of the reference substance and 20 microliters of the solution of the test sample respectively by adopting high performance liquid chromatography in the 2005 version of Chinese pharmacopoeia 2005 second part appendix VD; injecting the solution into a liquid chromatograph; and recording a chromatogram map; and (e) result judgment, namely nonassimilation is at a retention time of the chromatographic peak of vitamin D2. The method improves the quality control standard of medicinal accessories of the polysorbate 80, so that the interference of the related substances on the determination of the content of main medicament components and potential adverse reactions possibly caused by the related substances can be effectively avoided.