A study on the polysorbate 80 stability in various formulation buffers commonly used in biopharmaceuticals was performed, to investigate the excipients influence on polysorbate 80 degradation. Polysorbate 80 is a common excipient in biopharmaceutical products. However, its degradation will potentially impact the drug product quality, and may trigger protein aggregation and particles formation. Due to the heterogeneity of the polysorbates and the mutual effects with other formulation compositions, the study of polysorbate degradation is challenging. Herein, a real-time stability study was designed and performed. The polysorbate 80 degradation trend was monitored by fluorescence micelle-based assay (FMA), reversed-phase-ultra-performance liquid chromatography-evaporative light scattering detector (RP-UPLC-ELSD) assay, and LC-MS assay. These assays provide orthogonal results to reveal both the micelle-forming capability and the compositional changes of polysorbate 80 in different buffer systems. The degradation occurred after a period of storage under 25 °C in different trend, which indicates the excipients could impact the degradation kinetics. Upon comparison, the degradation is prone to happen in histidine buffer than in acetate, phosphate or citrate buffers. LC-MS confirms oxidation as an independent degradation pathway with detection of the oxidative aldehyde. Thus, it is necessary to pay more attention to the excipients selection and their potential impact on polysorbate 80 stability to achieve longer shelf life for the biopharmaceuticals. Besides, the protective roles of several additives were figured out, which could be applied as potential industrial solutions to the polysorbate 80 degradation issues.