Because syphilis is a public health concern, new strategies and tools for detecting active syphilis cases should be evaluated for future implementation. We assessed the laboratory performance of the DPP Syphilis Screen & Confirm rapid immunodiagnostic test (Chembio Diagnostics, Medford, NY, USA), using visual reading and the manufacturer’s electronic test microreader, for detection of treponemal and nontreponemal antibodies in 383 fully characterized stored serum specimens. We used the Treponema pallidum particle agglutination (TPPA) test and rapid plasma reagin (RPR) test as reference tests for the DPP Syphilis Screen & Confirm assay treponemal and nontreponemal components, respectively. The sensitivity values for treponemal antibody detection by electronic reader and visual interpretation were 83.2% and 85.9%, respectively, with 100% specificity. For nontreponemal antibody detection, the sensitivity values were 65.7% and 69.0% and the specificity values were 88.7% and 89.4% for electronic reader and visual interpretation, respectively. There was excellent correlation between visual interpretation and the microreader for either component (kappa coefficient, 0.953). When restricting the analysis to RPR titers of $1:8, the sensitivity was 96.9% for either reading method; numerical microreader values showed good correlation with RPR titers (Spearman rho of 0.77). The DPP Syphilis Screen & Confirm assay showed good performance, compared to reference syphilis tests, using serum. Field evaluation studies should be done to validate its use for detection of active cases and for monitoring of treated syphilis patients. IMPORTANCE Syphilis remains a public health problem; therefore, health systems must incorporate screening tools that allow a rapid and accurate diagnosis to provide adequate treatment. The DPP Syphilis Screen & Confirm Assay simultaneously detects treponemal and nontreponemal antibodies, emerging as an alternative for identifying cases in situations in which there is no infrastructure to perform conventional syphilis testing, but it is necessary to generate evidence regarding the performance of this technology in various scenarios. We found that the test performs well, compared to TPPA and RPR tests, using stored samples from participants at high risk of acquiring syphilis. Additionally, when the Chembio microreader was incorporated, similar results are obtained by the device, compared to those reported by trained laboratory professionals, and correlated with the semiquantitative results of the RPR test. We think that the use of the DPP Syphilis Screen & Confirm Assay with the microreader might help in detecting active syphilis cases and perhaps in monitoring treatment responses in the field.