We compared protein profiles of the synovial fluid of patients with rheumatoid arthritis and osteoarthritis by using surface-enhanced laser desorption/ionization mass spectrometry technology. With this approach, we identified a protein expressed specifically in the synovial fluid of the patients with rheumatoid arthritis. During the investigation, we found several reproducible and discriminatory biomarker candidates for distinction between rheumatoid arthritis and osteoarthritis. Among these candidates, a 10 850 Da protein peak was the clearest example of a single signal found specifically in the rheumatoid arthritis samples. This candidate was purified using a size-exclusion spin column followed by gel electrophoresis and subsequently identified by peptide mapping and post-source decay (PSD) analysis. The results clearly indicate that the protein is myeloid-related protein 8, which was verified by the enzyme immunoassay. It is known that the myeloid-related protein 8 level in serum and synovial fluid is related to disease activity in juvenile rheumatoid arthritis. The results suggest that the ProteinChip platform is useful to detect and identify protein biomarkers expressed specifically in diseases or in some stage of diseases.