Background: Lyme disease, caused by infection with Borrelia burgdorferi, is the most common vector-borne disease in the United States. The standard two-tier testing (STTT) algorithm suffers from low sensitivity, misinterpretation, and long turnaround time, preventing timely detection and treatment. To address these challenges, we hypothesized that the canine point-of-care (PoC) SNAP 4Dx Plus test used to detect Borrelia burgdorferi antibodies could be employed for human diagnosis. Materials and Methods: The SNAP 4Dx Plus testing was conducted in accordance with the manufacturer's instructions, with results read by manual inspection. All analyses were conducted using R version 4.3.1, and agreement between the PoC assay and the STTT was assessed using kappa statistics with GraphPad software. Results: We included 102 previously-tested human serum samples, of which 19 samples (18.6%) were STTT positive. Compared to the STTT, the SNAP 4Dx Plus test demonstrated a low sensitivity of 0.16 (95% CI 0.03 to 0.40). Conclusion: Overall, our results do not support the use of the SNAP 4Dx Plus LD assay for the diagnosis of human Lyme disease. Differences in antibody concentrations between human and canine samples may partly explain our findings.