Rheumatoid arthritis (RA) is a chronic and systemic inflammatory disorder that primarily affects the flexible joints and may also affect a number of tissues and organs. The progression of RA involves an inflammatory response of the capsule around the joint, swelling of synovial cells with excess synovial fluid (SF), and the development of fibrous tissue in the synovium. Since the progressive pathology of the disease often leads to the irreversible destruction of articular cartilage and ankylosis of the joint, early diagnosis of RA is essential. Thus, we undertook a comparative proteomic approach to investigate novel biomarkers for early diagnosis using SFs and serums from RA patients. As a result, we identified 32 differentially expressed spots in SFs and 34 spots in serums. The differential expression of the STEAP4 and ZNF 658 proteins were validated using immunoblotting of the SFs and serums, respectively. These data suggest that differentially expressed proteins in SFs and serums could be used as RA-specific biomarkers for the diagnosis and monitoring of RA. Furthermore, these findings advance our understanding of the molecular etiopathogenesis of RA.