The purpose of this work was to study the mechanistic pathways of degradation of polysorbates (PS) 20 and PS80 in parenteral formulations. The fate of PS in typical protein formulations was monitored and analyzed by a variety of methods, including 1H NMR, high-performance liquid chromatography/evaporative light scattering detection, and ultraviolet–visible spectroscopy. Oxidative degradation of PS in neat raw material was studied using thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and headspace gas chromatography–mass spectrometry. TGA–DSC studies revealed that autoxidation via a radical mechanism is dominated by statistical random scission in PS20 and PS80. Thermal initiation of radical formation occurs at the polyoxyethylene (POE) as well as the olefin sites. In PS80, radical initiation at the olefinic site precedes initiation at the POE site, leading to modified degradation profile. Corresponding to these results, in aqueous formulations, a surge peroxide content was detected in PS20-containing samples and in higher concentrations in those containing PS80. Hydrolysis in aqueous formulations, as followed by 1H NMR, was found to have a half-life of 5months at 40°C. On the basis of the obtained results, PSs degrade mainly via autoxidation and also via hydrolysis at higher temperatures. Further studies are required to investigate on potential effects of degradation on surface activity and protein stability in PS-containing formulations.