Evaluation of immobilized-proteins on bio-devices is important for the development of sophisticated devices. Lysozyme molecules immobilized on substrates were evaluated by means of time-of-flight secondary ion mass spectrometry (TOF-SIMS). Two types of the lysozyme-immobilized samples were prepared by controlling the binding parts, i.e., the amino groups or carboxyl groups, of the protein. The TOF-SIMS spectra of each sample were analyzed with mutual information to select fragment ions specific to each sample. According to the results, differences between the samples being immobilized in the different ways are suggested, and the surface structure of the lysozyme molecule immobilized at amino groups is determined based on three-dimensional structure of lysozyme in the Protein Data Bank. (c) 2008 Elsevier B.V. All rights reserved.