Abstract Polysorbates (also known as “Tween”) are common components of protein formulations used to minimize protein adsorption and stabilize the protein. These nonionic surfactants are heterogenous mixtures of fatty acids with a complex reversed-phase profile due to the inhomogeneity of the polymers present. Polysorbates can be oxidized, which can be hard to detect in the complex polymer profile. Further adding to the analytical challenge is the lack of a chromophore for the detection of these polymers. The routine analysis of polysorbates in protein formulations was greatly improved through the introduction of online solid-phase extraction (SPE) to simplify the polysorbate profile for quantification. However, this method combines many of the polysorbate polymers into a single peak for detection, thus limiting its effectiveness for detecting degradation. To address the need for a stability indicating method without the complexity of the reversed-phase profile, an optimized online SPE method was developed and investigated. Using polysorbate 80, this investigation shows that further expanding the step gradient can yield a profile that is stability indicating and available for routine testing of protein formulation.