ABSTRACT: Zika virus (ZIKV) is a mosquito-borne flavivirus that emerged recently as a global health threat, causing a pandemic in the Americas. ZIKV infection mostly causes mild disease, but is linked to devastating congenital birth defects and Guillain-Barré syndrome in adults. The high level of cross-reactivity among flaviviruses and their cocirculation has complicated serological approaches to differentially detect ZIKV and dengue virus (DENV) infections, accentuating the urgent need for a specific and sensitive serological test. We previously generated a ZIKV nonstructural protein 1 (NS1)-specific human monoclonal antibody, which we used to develop an NS1-based competition ELISA. Well-characterized samples from RT-PCR-confirmed patients with Zika and individuals exposed to other flavivirus infections or vaccination were used in a comprehensive analysis to determine the sensitivity and specificity of the NS1 blockade-of-binding (BOB) assay, which was established in laboratories in five countries (Nicaragua, Brazil, Italy, United Kingdom, and Switzerland). Of 158 sera/plasma from RT-PCR-confirmed ZIKV infections, 145 (91.8%) yielded greater than 50% inhibition. Of 171 patients with primary or secondary DENV infections, 152 (88.9%) scored negative. When the control group was extended to patients infected by other flaviviruses, other viruses, or healthy donors (n = 540), the specificity was 95.9%. We also analyzed longitudinal samples from DENV-immune and DENV-naive ZIKV infections and found inhibition was achieved within 10 d postonset of illness and maintained over time. Thus, the Zika NS1 BOB assay is sensitive, specific, robust, simple, low-cost, and accessible, and can detect recent and past ZIKV infections for surveillance, seroprevalence studies, and intervention trials.