Rheumatoid arthritis (RA) is a persistent autoimmune disease stimulated by a sophisticated system of proinflammatory cytokines such as TNF-α and IL -17. They contribute significantly to the maintenance of inflammation and mediation of joint damage. The current study aimed to examine the relationship between serum concentrations of TNF-α, IL-17 A, and IL-17 F and disease activity in Yemeni RA patients. A case-control study was carried out on 101 RA patients and 80 healthy controls (HCs) matched for age and sex. The serum concentration of TNF-α, IL-17 A, and IL-17 F was determined through an enzyme-linked immunosorbent assay (ELISA). Other hematological, inflammatory parameters and serological tests were also assessed. TNF-α, IL-17 A and IL-17 F serum levels were significantly elevated in RA patients than in HCs (P < 0.001) and had a positive correlation with disease activity in RA groups (P < 0.001). Furthermore, a significant positive correlation between levels of TNF-α, IL-17 A, and IL-17 F and the DAS28. The ROC analysis revealed that all the three cytokines TNF-α, IL-17 A, and IL-17 F were effective in distinguishing between RA patients and HCs, with TNF-α having the highest diagnostic accuracy. To define moderate to high disease activity, IL-17 A demonstrated the best individual quality, whereas TNF-α and IL-17 F demonstrated moderate quality. Interaction of the cytokines improved the diagnostic value as both TNF-α and IL-17 A combination and triple combination of TNF-α, IL-17 A and IL17F which supports the benefits of multiplex cytokine testing in RA activity testing as well as discrimination of moderate to high activity RA.