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The lack of specific clinical symptoms for patients in the early stage of rheumatoid arthritis (RA) has created strong interest in the laboratory diagnosis of RA. The main laboratory markers of RA, rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPAs), can be found in patients with other pathologies and in healthy donors. Even today, there is no single laboratory test that can diagnosis RA with high sensitivity and specificity. To improve the diagnosis and treatment of RA, alternative biomarkers, including 14-3-3η protein, connective tissue growth factor (CTGF), antibodies against PAD4, antibodies against BRAF, and anti-acetylated and anti-carbamylated protein antibodies have been studied extensively. The use of a multiple biomarker approach, the simultaneous measurement of a set of biomarkers, is an alternative strategy for the diagnosis of RA and for predicting the therapeutic effect of biological disease-modifying antirheumatic drugs (DMARDs). However, despite the large number of studies, only a few biomarker combinations have been validated and can be applied in clinical practice. In this article, results of studies focused on the multiple biomarker approach (both multiplex and combined single-analyte assays) to diagnose RA and to predict response to biological drug therapy are reviewed. Additionally, general factors limiting the use of multiplex analysis in RA diagnostics and therapy are discussed.