An anti-Zaire Ebola virus (EBOV) glycoprotein (GP) immunoglobulin G (IgG) enzyme linked immunosorbent assay (ELISA) was developed to quantify the serum levels of anti-EBOV IgG in human and non-human primate (NHP) serum following vaccination and/or exposure to EBOV. This method was validated for testing human serum samples as previously reported. However, for direct immunobridging comparability between humans and NHPs, additional testing was warranted. First, method feasibility experiments were performed to assess cross-species reactivity and parallelism between human and NHP serum samples. During these preliminary assessments, the goat anti-human IgG secondary antibody conjugate used in the previous human validation was found to be favorably cross-reactive with NHP samples when tested at the same concentrations previously used in the validated assay for human sample testing. Further, NHP serum samples diluted in parallel with human serum when tested side-by-side in the ELISA. A subsequent NHP matrix qualification and partial validation in the anti-GP IgG ELISA were performed based on ICH and FDA guidance, to characterize assay performance for NHP test samples and supplement the previous validation for human sample testing. Based on our assessments, the anti-EBOV GP IgG ELISA method is considered suitable for the intended use of testing with both human and NHP serum samples in the same assay for immunobridging purposes.