The non-Treponemal tests such as Rapid Plasma Reagin test (RPR) or the Venereal Disease Reference Laboratory test are the most commonly used test for screening of syphilis in the blood centers in India. Now, with the availability of Enzyme-linked immunosorbent assay (ELISA) and Immunochromatographic assays in the market, we decided to evaluate these assays in comparison with Treponema pallidum Haemagglutination Assay (TPHA) which was considered as a gold standard for this study. A total of 8 685 samples of voluntary blood donors were tested on Trepolisa 3.0 and then the initially reactive samples were retested in duplicate on the same assay as well as on Omega Pathozyme, RPR, RAPHA (Rapid Anti-Treponema pallidum Assay), and TPHA. Of the 158 initially reactive samples, 104 were repeatedly reactive on the same assay, 85 were reactive with RPR, 77 were reactive with RAPHA, 60 were reactive on Omega, and 53 were confirmed reactive on TPHA. 48 (56.4%) of the results on RPR were biological false positive, while 21.9% of results were false negative on RPR. We evaluated that Omega Pathozyme was quite in agreement with TPHA as compared with Trepolisa 3.0, RAPHA, and RPR. We concluded that Omega Pathozyme (ELISA) can be considered as a suitable test for screening of syphilis in a blood center.