Biopharmaceuticals, also known as biologics, are the fastest growing segment in the drug development market. However, biologic-based formulations are inherently unstable and require additives such as surfactants to prevent denaturation. This work focused on Tween 20 and Tween 80, which are the most commonly used surfactants, across two different levels of purity and different concentrations to try and develop an enhanced understanding of biologic-polysorbate interactions. Three model proteins were selected with a range of sizes and amino acid compositions. Multiple analytical techniques were employed, namely UV-visible spectroscopy (UV-vis), size-exclusion chromatography (SEC), isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC) and molecular docking. A newly developed method of UV-vis analysis alongside SEC indicated that at low concentrations (below the critical micelle concentration (CMC)) none of the surfactants changed the shape of the model proteins or the extent of aggregation. However, once concentrations rose above the CMC, exposure to the surfactants produced changes to protein conformation. ITC confirmed that surfactants underwent binding-like activity with two of the three model proteins. DSC demonstrated that the addition of surfactants can raise the thermal stability of proteins and prevent denaturation. Molecular docking results indicated the presence of hydrophobic interactions between proteins and polysorbates. Finally, this study investigated the effects of polysorbates on protein-peptide interactions, whereby polysorbates directly affected the binding affinity of proteins and peptides. Furthermore, adding polysorbates to a protein-peptide formulation can prevent denaturation of proteins by interfacial protection but do not convey increased thermal stability when compared with protein-peptide alone.