Background Syphilis causes up to 1,500,000 congenital syphilis cases annually. These could be prevented if all pregnant women were screened, and those with syphilis treated with a single dose of penicillin before 28 weeks gestation. In recent years, rapid point-of-care tests have allowed greater access to syphilis screening, especially in rural or remote areas, but the lack of quality assurance of rapid testing has been a concern. We determined the feasibility of using dried blood spots (DBS) as specimens for quality assurance of syphilis serological assays. Methods We developed DBS extraction protocols for use with Treponema pallidum particle agglutination assay (TPPA), Treponema pallidum haemagglutination assay (TPHA) and an enzyme immunoassay (EIA) and compared the results with those using matching plasma samples from the same patient. Results Since DBS samples showed poor performance with TPHA and EIA (TPHA sensitivity was 50.5% (95% confidence interval: 39.9–61.2%) and EIA specificity was 50.4% (95% CI: 43.7–57.1%), only the DBS TPPA was used in the final evaluation. DBS TPPA showed an sensitivity of 95.5% (95% CI: 91.3–98.0%) and a specificity of 99.0% (95% CI: 98.1–99.5%) compared to TPPA using plasma samples as a reference. Conclusion DBS samples can be recommended for use with TPPA, and may be of value for external quality assurance of point-of-care syphilis testing.