Abstract: There is evidence in the literature that silicone oil, a lubricant, can induce aggregation in protein formulations delivered through prefilled syringes. Surfactants are commonly used to minimize protein–silicone oil and protein–container interactions; however, these interactions are not well characterized and understood. The purpose of this manuscript was to understand the competitive interactions of a fusion protein with the silicone oil in the presence of Tween® 20. An adsorption isotherm for Tween® 20 at the silicone oil/water interface, using silicone oil coated quartz crystals, was generated at 25°C to identify surface saturation concentrations. A concentration of Tween® 20 providing interfacial saturation was selected for protein adsorption studies at the silicone oil/water interface. The surfactant molecules adsorbed at the interface in a monolayer with a reduced viscoelastic character in comparison to the bound protein layer. A significant reduction in protein adsorption was observed when the surfactant was present at the interface. No desorption of the pre-adsorbed protein molecules was observed when Tween® 20 was introduced, suggesting that the protein has strong interactions with the interface. However, both, Tween® 20 and protein, adsorbed to the silicone oil/water interface when adsorption was carried out from a mixture of protein and Tween® 20. [Copyright &y& Elsevier]