Polysorbate 80 is an important excipient in therapeutic monoclonal antibody drugs. Excessive use can lead to hemolysis and other adverse consequences. Therefore, its content should be strictly controlled during drug release for safety considerations. Colorimetric methods, gas chromatography, and liquid chromatography are most commonly used for analysis. Colorimetric methods are gradually being replaced by other methods due to their poor sensitivity and accuracy. The preliminary treatment step for gas chromatography is complex and time-consuming. Liquid chromatography has been used for traditional Chinese medicine and chemical drugs. Here, we established a method to determine the content of polysorbate 80 in monoclonal antibodies and verified the methodology. The results show that the method has high specificity, good accuracy, good precision, and reasonable robustness. It can determine the polysorbate 80 content in therapeutic monoclonal antibody drugs.