We evaluated the utility of 2 types of commercially available antigens as positive controls in the Rapid Analyte Measurement Platform (RAMP®) West Nile virus (WNV) assay. Purified recombinant WNV envelope antigens and whole killed virus antigens produced positive RAMP results and either type would be useful as a positive control. Killed virus antigens provide operational and economic advantages and we recommend their use over purified recombinant antigens. We also offer practical applications for RAMP positive controls and recommendations for preparing them.