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ABSTRACT Syphilis is a multistage chronic sexually transmitted infection caused by the spirochete Treponema pallidum subsp. pallidum (T. pallidum). Today, syphilis is still endemic in low- and middle-income nations and has become resurgent in many high-income countries. Infection can lead to severe sequelae in untreated patients, while vertical transmission of T. pallidum is associated with high stillbirth rates and neonatal death. The combination of non-treponemal and treponemal tests allows syphilis serodiagnosis and monitoring of treatment response, albeit not without limitations. In this work, we developed a minimal array for multiple parallel detection of antibodies specific to the immunodominant T. pallidum antigens Tp0435 and Tp0574 and 14 additional proteins known to be immunogenic during infection but only partially or not at all evaluated for their diagnostic potential. To assess whether reactivity to these antigens could improve early syphilis diagnosis, serve as biomarkers for disease staging, and monitor response to treatment, we tested the array using 217 serum specimens longitudinally collected pre- and post-treatment from 120 syphilis patients. Results showed significantly different reactivity to a subset of antigens in pre-treatment sera (baseline) when covariates such as syphilis stage, syphilis history, and HIV status were factored in. Furthermore, reactivity to several antigens significantly decreased in post-treatment sera compared to baseline. Although studies with larger sample panels will be needed to validate these findings, this study supports the screening of T. pallidum proteomic arrays to identify antigens that could serve as predictors of response and antigens that could help with early syphilis diagnosis. IMPORTANCE This manuscript explores the host humoral response to selected antigens of the syphilis agent during infection to evaluate their potential use as diagnostic tests and markers for treatment.