We demonstrate the application of time-of-flight secondary ion mass spectrometry (TOF-SIMS) in conjunction with multivariate statistics to differentiate trace levels of denatured proteins in adsorbed monolayers; specifically, human serum albumin (HSA) on oxidized silicon substrates. Subtle differences in protein conformation due to thermal denaturation of HSA, unable to be determined by dynamic light scattering nor circular dichroism, were differentiated by TOF-SIMS. The fragmentation pattern is highly sensitive to protein conformation, allowing assessment of relative amounts of proteins in mixtures and quantifying amounts of denatured protein in a sample. Discussion is presented on ascribing orientation and conformational differences between samples based upon TOF-SIMS spectra. This has implications for detecting denatured protein in biotechnology and medical applications.