There are multiple serological testing methods and vaccines using different Borrelia burgdorferi antigens. Previous studies have demonstrated that different outer surface proteins (Osps) can serve as antigens that can be detected by antibody testing. These Osps, as well as the C6 peptide, serve as markers of early natural infections, late natural infections, and vaccine exposure. Many antigen preparations used in Lyme testing do not differentiate between natural exposure vs vaccine-induced antibodies. The primary goal of this study was to optimize detection of serum antibodies against multiple natural and artificial peptides of B burgdorferi for use on the Accuplex4 BioCD system platform (Antech Diagnostics). The secondary goal was to apply the assay to sera from dogs exposed to B burgdorferi-infected Ixodes scapularis before or after vaccination with 1 of 3 vaccines. The test was able to differentiate between antigens found in naturally exposed animals vs those exposed via vaccination. Because this method tests multiple types of antibodies observed at different stages of infection, the assay could be used to refine the assessment of the timing of B burgdorferi exposure or vaccination status. Funding for this study was provided by Antech Diagnostics.