During the period from May to November 2015, Zika virus (ZIKV) infections gained global attention in Brazil. ZIKV, a Flavivirus transmitted by Aedes mosquitoes, initially identified in 1947, has evolved from a historically inconspicuous pathogen to a significant public health concern. Beyond mild symptoms resembling dengue fever, severe conditions such as microcephaly and Guillain-Barré syndrome have been linked to ZIKV. This study focuses on epitope mapping in ZIKV non-structural proteins (NS1, NS3, and NS5), exploring the interaction with maternal and newborn immune responses through SPOT-synthesis. The objectives include establishing epitope profiles in Zika-positive individuals and correlating maternal-newborn responses. Serum samples were collected from mothers and newborns, was born in Zika Cohort Jundiai research, and Sergipe Cohort. Epitope mapping experiments was conducted using a cellulose membrane-based SPOT-synthesis technique. Notably, regions in NS1, NS3, and NS5 proteins exhibited high membrane reactivity and epitope recognition. NS1, a key antigenic marker, holds potential as an early biomarker for ZIKV detection. Additionally, NS3 and NS5 proteins displayed immunogenic potential with differential responses in newborns. The study enhances understanding of ZIKV immunology and suggests further investigations for epitope validation and potential vaccine development.