In the fabrication and interfacial analysis of novel biomaterials and their biological interactions, vacuum-based methods occupy a prominent role. Much research centers on the fabrication of "hybrid" biomaterials, which comprise a synthetic carrier material and an immobilized layer of biologically active molecules. Low temperature gas plasma methods are well suited to the attachment of reactive chemical groups onto polymers. Alternatively, plasma polymer interlayers can be used to provide reactive surface groups for the covalent interfacial immobilization of proteins. Vacuum-based surface analysis techniques characterize the surface properties of a material and increasingly are applied to the study of interfacial interactions with biological molecules. In this talk I will present examples of recent work on the immobilization of proteins and synthetic peptides on polymers via plasma polymer interfacial bonding layers. Detailed, multitechnique characterization of surface derivatizations and protein immobilizations is essential since proteins can adsorb and thereby mimic an intended covalent immobilization. Intended attachments are first modelled using derivatization reactions, and the surface density of reactive groups is thus determined. MALDI mass spectrometry is uniquely suited to the detection of adsorbed biomolecules at amounts much below monolayer coverage, and this method has been used to distinguish between covalent and adsorptive immobilizations. MALDI-MS is also eminently suited to the study of which proteins adsorb from complex, multicomponent media. For instance, the ways in which different surface chemistries of contact lenses influence which proteins adsorb onto lenses worn by human volunteers, has been characterized by MALDI-MS, and this information is being used for the guided design of improved coatings. Finally, I will discuss how AFM in the force mode provides complementary information to vacuum-based analysis methods.