Combining the detection of syphilis and HIV antibodies into one point-of-care test integrates syphilis screening into already existing HIV screening programs, which may be particularly beneficial in settings such as antenatal care. Using the INSTI Multiplex downward-flow immunoassay, we tested 200 stored serum samples from high-risk patients enrolled in a longitudinal study on HIV infection and syphilis in Peruvian men who have sex with men and transgender women. This rapid assay detected HIV and Treponema pallidum serum antibodies with sensitivities of 100% (95% confidence interval [CI], 95.9% to 100%) and 87.4% (95% CI, 81.4% to 92.0%), respectively, and specificities of 95.5% (95% CI, 89.9% to 98.5%) and 97.0% (95% CI, 84.2% to 99.9%), respectively (n200). The sensitivity for syphilis antibody detection was higher in patients with a rapid plasma reagin titer of>1:8 (97.3%) than in those with a titer of<1:4 (90%) or a nonreactive titer (66.7%).