Techniques for patterned modification of substrate surfaces are important for forming microarrays on protein chips. A hexamethyldisiloxane plasma-polymerized film (HMDS PPF) was deposited on a glass substrate and the resulting surface was partially modified by subsequent nitrogen plasma treatment with a patterned shadow mask. When surface adsorption of an antibody protein (F(ab')2 fragment of anti-human immunoglobulin G) was visualized by fluorescence microscopy, distinct 8080 µm2 square spots were observed, surrounded by a non-fluorescent 80 µm-wide grid. This pattern could be attributed to proteins selectively adsorbed onto the nitrogen plasma-treated regions but not onto the surface of pristine HMDS PPF. This provided a simple fabrication method of protein patterning.