Membrane proteins (MPs) are critical contact points for transducing environmental cues across cell membranes and hence have become major pharmaceutical targets. Despite recent advances in structural biology, elucidation of MP function and mechanisms remains challenging partly because structures only represent static snapshots at the endpoints of protein dynamics, while the higher complexity of MPs requires exploration of the full energy landscape. My thesis aims to advance our understanding of MP mechanisms by non-invasively probing MP dynamics in solution using hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS). The main project details investigation of the thermodynamics and dynamics of prestin, whose voltage-dependent conformational changes underlie the acute hearing sensitivity for mammals. We find that the conformational stability of the helices associated with the anion-binding site accounts, in large part, for prestin’s distinct differentiation from its transporter-evolutionary origin. Our results argue that the folding equilibrium of the anion-binding site plays a novel and central role in prestin’s voltage-sensing mechanism and electromotility. Additionally, we present the development and application of in vivo HDX-MS to BtuB, a TonB-dependent B12 transporter found in E. coli outer membranes. HDX-MS measured in living E. coli cells showed that B12 alone is sufficient to break a critical salt bridge in BtuB, leading to the unfolding of the amino terminus for TonB binding. The ability to measure HDX in vivo is generalizable which opens up a wide range of HDX studies on proteins in their native context. Overall, our studies on prestin have deepened our understanding of mammalian hearing sensation. We have significantly advanced the utility of HDX-MS to in vivo systems and MPs embedded in lipid bilayers. This work on HDX-MS and MPs have advanced our knowledge of MP functions and mechanisms from a distinct thermodynamic perspective.