Human (h)IgG2 monoclonal antibodies (mAbs) are potent agonists due, in part, to their ability to undergo disulfide shuffling within their hinge regions. Herein, we describe a rapid, sensitive, collision-induced unfolding (CIU) assay that possesses a predictive relationship between gas-phase protein unfolding and agonism in hIgG2 variants. Furthermore, our results highlight the significance of hinge engineering in tuning mAb structure-function relationships for the development of future biotherapeutics.