To support the rapid development of an antibody cocktail against Ebola virus and avoid unnecessary exposure to infectious environments, an automatic and fast turnover triplex assay was developed using Simoa (Quanterix Corporation, MA, USA). A robust triplex assay was developed and validated for simultaneous quantification of the antibody cocktail against Ebola virus in cynomolgus serum. The assay had a quantitation range of 78.1-5000 ng/ml. The intra- and interassay precisions (%CV) were within 11.4 and 13.9%, and the accuracies (%RE) were within -10.8 to 6.8%, respectively. Cross-reactivity was evaluated, and the results met the acceptance criteria. The assay was successfully applied to a pharmacokinetics study following a single-dose intravenous administration of 10 mg/kg the antibody cocktail against Ebola virus to cynomolgus monkeys.