The adsorption of proteins at biomaterial surfaces depends on the properties of the substrate and can cause changes in protein conformation. Time-of-flight secondary ion mass spectroscopy (ToF-SIMS) was used in this study to characterize human serum albumin (HSA) adsorption on two different polyearbonate surfaces: a native membrane and a hydrophilic treated one. The amount adsorbed as a function of HSA concentration in solution was compared for the two substrates. The treated membrane was found to have a lower affinity for albumin than the native one. Principal component analysis was used to reveal changes in albumin conformation as a function of albumin concentration in solution and to compare the conformations adopted on the two substrates. The albumin conformation was different on the two substrates, and in every case, the protein lost its native structure. A correlation was found between the amount adsorbed on the hydrophilic surface and the albumin conformation on this surface.