The characterization of adsorbed protein films with ultrahigh vacuum (UHV) surface analysis techniques requires dehydration of the samples, which can cause significant alterations in protein conformation. Trehalose coating was used in this study to inhibit these conformational changes from occurring when preparing samples for analysis in UHV. Surface plasmon resonance (SPR) analysis showed that air-dried films of trehalose-stabilized antiferritin and anti-IgM both retained a significant proportion of their hydrated antigen binding activity. In contrast, air-drying without trehalose protection resulted in the adsorbed protein films losing most of their antigen binding activity. Structural differences between trehalose-stabilized and unstabilized protein films were then analyzed with static time-of-flight secondary ion mass spectrometry (ToF-SIMS). By application of principle component analysis (PCA) to the static ToF-SIMS spectra, the biological activity difference observed in SPR was correlated to changes in protein conformation. Trehalose-protected proteins retained a greater degree of their original conformation than the unprotected proteins. This suggests coating adsorbed protein films with trehalose prior to air-drying and introduction into UHV allows ToF-SIMS to analyze adsorbed proteins in a state that is more representative of their actual structure in an aqueous environment.