The aim was to compare the sensitivity of different grades of polysorbate 20 (PS20) and polysorbate 80 (PS80) against enzymatic hydrolysis and oxidative degradation in pharmaceutically relevant buffer systems. For this purpose, a fast liquid chromatography charged aerosol detection method was developed which allows to (1) differentiate between hydrolytic and oxidative PS degradation and (2) to monitor the PS decay over time. Systematic enzymatic and oxidative forced degradation studies were conducted with multicompendial PS20 and PS80, as well as all-laurate PS20 and all-oleate PS80 (with >98% oleic acid, as required by the Chinese Pharmacopoiea since 2015). No differences in the sensitivity toward enzymatic degradation were observed between multicompendial PS and high purity grade PS. However, all-laurate PS20 and all-oleate PS80 have a higher predisposition for oxidative degradation as compared to multicompendial PS20 and PS80. The buffer system used within the study played thereby a key role: histidine showed a protective effect against hydrogen peroxide-induced oxidation, whereas hydrogen peroxide oxidation of PS in acetate buffer was severe under the experimental conditions. Furthermore, ethylenediaminetetraacetic acid protected PS20 and PS80 against oxidative degradation in histidine buffer.