Significance Zika virus (ZIKV), a mosquito-borne flavivirus with homology to dengue virus (DENV), has become a public health threat, particularly because of its association with severe congenital birth defects. The high level of cross-reactivity among flaviviruses and their cocirculation has complicated serological approaches to detect ZIKV infections. Thus, there is an urgent need for a specific serological assay to discriminate ZIKV infection from other flaviviruses. This study demonstrates that the antibody-based assay we developed and implemented in five countries has high specificity and sensitivity in the detection of recent and past ZIKV infections. The ZIKV nonstructural protein 1 (NS1) blockade-of-binding ELISA assay is a simple, robust, and low-cost solution for Zika surveillance programs, seroprevalence studies, and intervention trials in flavivirus-endemic areas.