Orientation of immobilized proteins on bio-devices is important to obtain their high performance. Structural change in a particular area of a protein is also very important for the study of the protein performance and the evaluation of the bio-devices. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is able to analyze upper surface of one layer of molecules. Orientation of immobilized proteins can be evaluated based on determination of a partial structure, representing ensemble of amino acids, on the surface part. The model protein, bovine serum albumin (BSA), is immobilized on the substrate, indium-tin oxide (ITO) glass electrode, by covalent bonding. Two types of the oriented samples were prepared by controlling the binding parts of BSA. The results from TOF-SIMS spectra analysis were compared to the amino acid sequence to examine surface parts of the immobilized BSA at different groups. The surface parts of the BSA molecules immobilized on ITO glass plates with different parts of the molecule, amino groups or carboxyl groups, are evaluated by means of TOF-SIMS. The orientation differences were clearly shown in TOF-SIMS spectra of the samples. Furthermore, fragment-ion-generating parts of immobilized-BSA are determined by fragment ions of particular combinations of amino acids in the sequence of BSA. [DOI: 10.1380/ejssnt.2006.614]