Background: Borrelia burgdorferi sensu stricto is the causative agent of Lyme disease (LD). Possible early symptoms include flu-like symptoms and erythema migrans and later, the risk of disruption of the nervous system, joints, and heart. A two-tiered testing method is employed for serological diagnostics. The Public Health Agency of Canada guidelines recommend that samples tested negative on first-tiered test need not be confirmed by second-tiered test. Due to the challenging nature of diagnosis leading to misconceptions among physicians about false negatives, confirmatory testing is requested despite the initial negative result. Methods: Hundred screen-negative Lyme patient samples from 2007 to 2016 were tested by Western blot (WB) second-tiered confirmatory test upon physician's request in British Columbia to study the first-tiered screening test sufficiency. Results: Those negative for first-tiered enzyme-linked immunosorbent assay were also negative by WB. Conclusion: Results demonstrate that confirmatory testing is not necessary on screen-negative samples. Hence, first-tiered test is sufficient to rule out LD.