To assess the ability of the assay to distinguish infections by DENV and Asian ZIKV, we performed DENV and ZIKV homologous and heterologous LFn-NS3 protease and helicase stimulation of date-matched late-convalescent-phase peripheral blood mononuclear cells (PBMCs) in separate gamma interferon (IFN-γ) and tumor necrosis alpha (TNF-α) ELISPOT tests among the serological-validated pDENV, pZIKV, sDENV, ZIKVwpDENV, and undetermined cases. The gene discussed is IFNG; the disease is infection.