By taking advantage of their unique difference in hydration sensitivity, we have shown that dual polarization interferometer (DPI) and quartz-crystal microbalance with dissipation monitoring (QCM-D) measurements can be used together to explore the degree of desolvation involved in the binding of small drug molecules to an immobilized bovine serum albumin film in real time. Results with DPI and QCM-D show significantly different mass values for three ligands of varying hydrophobicities that may be attributed to changes in the degree of hydration of the ligand-protein complexes in accordance with the physicochemical properties of the ligands. Furthermore, our data suggest that masses measured by QCM-D can be overwhelmed by changes in water content of ligand-protein, binary complexes, which has important consequences for future studies using mechanical resonators to study protein-binding events.