The goal of the present study was to determine performance of two traditional non-treponemal tests for syphilis. Syphilis sera (n = 209) included different stages of cases, and control sera (n = 247) were from patients with tumors, leprosy, systemic lupus erythematosus, hepatitis, pregnant women and healthy individuals. ELISA, Treponema pallidum particle agglutination and rapid treponema-specific tests were used as gold standards. Rapid plasma reagin or toluidine red unheated serum test had a sensitivity and specificity of over 95%. False-negative reactions of rapid plasma reagin and toluidine red unheated serum test were observed majorly in primary and latent syphilis cases, and false-positive reactions were present in systemic lupus erythematosus, hepatitis-infected patients. Overall, both non-treponemal tests had high sensitivities and specificities making the assays attractive as screening tests for syphilis. When examined on a WHO reference serum sample and based on lower limit of detection, non-treponemal tests were less sensitive than treponema-specific tests.