Protein aggregation remains a major challenge in biopharmaceutical development, despite extensive efforts to mitigate this process. Mechanical stresses, such as shaking, promote aggregation through interface-mediated pathways. During shaking, proteins are simultaneously exposed to three types of interfaces, air–liquid, solid–liquid, and air–liquid–solid; however, the relative contribution of each interface to aggregation remains unresolved. In this report, we systematically evaluated the disruption of protein aggregates at the glass vial surface by shear flow generated during shaking. Computational fluid dynamics was used to estimate shear rates at the vial surface under shaking conditions that increase the amount of protein aggregates. The shear rate, along with a harsher shear condition, was then applied to protein-coated glass vial surfaces using a gyroscopic mixer. Importantly, the vials were filled with buffer solution to eliminate contributions from the air–liquid and air–liquid–solid interfaces. Under these controlled conditions, no increase in protein aggregation was detected, indicating that shear-mediated disruption of protein films by shear flow at the solid–liquid interface plays only a minor role in protein aggregation. Complementary shaking experiments with headspace showed no measurable aggregation attributable to the air–liquid–solid interface. These findings identify the air–liquid interface as the dominant contributor to protein aggregation during shaking. This study clarifies the mechanistic role of individual interfaces in shake-induced protein aggregation and offers practical guidance for formulation design, container-filling strategies, and process conditions to minimize protein aggregation in biopharmaceutical products.