Rönnberg B, et al. - Clinicians here developed a novel “pan-Flavi” suspension multiplex immunoassay (PFSMIA) using 25 antigens, whole virus (WV), non-structural protein 1 (NS1), and envelope (E) proteins, from 7 zoonotic flaviviruses for specific detection of Zika virus (ZIKV) and Dengue virus (DENV) IgM and IgG. In this work, PFSMIA provided sensitive and specific ZIKV and DENV serology, supporting high-throughput serological surveillance and a minimized need for laborious and expensive virus neutralization assays.