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Abstract : Introduction: Syphilis, the first transfusion-transmitted disease described, was reported commonly before 1950. Screening of blood donors have been performed with serological test from the early of stages of blood transfusion practices.. Although the risk the transmission of syphilis through blood is negligible, in India, it is mandatory that blood banks screen every donation for syphilis3. The study was performed to evaluate the true seroprevalence of syphilis in blood donors as well as to evaluate the suitability of Immunochromatographic test, PaGIA, TPHA and ELISA as replacement for RPR either for screening or confirmation of syphilis in blood donors. Material & Methods: The total number of donors who were screened for syphilis were 28,544. All the donors were screened for HIV-1, 2, Hepatitis B, Hepatitis C, syphilis and malaria. Rapid plasma reagin (RPR) method was done as a primary screening method of syphilis using carbogen particles (Tulip laboratories). A total of 132 RPR positive and 132 RPR negative control sera were included in the study to compare the additional test performed. Results: Out of the total samples tested , 132 donors were found to be RPR reactive with 0.46% seropositivity. The seroprevalence was 0.46% (n= 127) among male donors and 0.43% (n =05) in female donors which was not statistically significantly different (p>0.05). The sensitivity, specificity, PPV, and NPV of treponemal tests was done and it was seen that PaGIA, ICT, and ELISA had sensitivity of 100%, 98.8% and 100%, respectively and the specificity of PaGIA, ICT, and ELISA was found to be 78.7%, 97.8%, and 93.5%. An overall agreement of 91.6 % was found in all four treponemal tests. Conclusion: On comparing the RPR titer with other treponemal tests performed, it was found that titer >16 gave the good prediction of positive treponemal tests. Other studies have also shown correlation of RPR or VDRL titre with treponemal tests. In our study, the sensitivity of ELISA, TPHA and PaGIA were similar and comparable. The positive predictive value and negative predictive values for PaGIA were 89.4% and 100%, respectively. In our study, treponemal assay had higher specificity than cardiolipin assay. The number of false positive samples can be reduced by using a method for screening which has higher specificity.