Lyme disease (LD) diagnosis using the current two-tier algorithm is constrained by low sensitivity for early-stage infection and ambiguity in determining treatment response. We recently developed a protein microarray biochip that measures diagnostic serum antibody targets using grating-coupled fluorescent plasmonics (GC-FP) technology. This strategy requires microliters of blood serum to enable multiplexed biomarker screening on a compact surface and generates quantitative results that can be further processed for diagnostic scoring. The GC-FP biochip was used to detect serum antibodies in patients with active and convalescent LD, as well as various negative controls. We hypothesized that the quantitative, high-sensitivity attributes of the GC-FP approach permit: 1) screening of antibody targets predictive for LD status, and 2) development a diagnostic algorithm that is more sensitive, specific, and informative than the standard ELISA and Western blot assays. Notably, our findings led to a diagnostic algorithm that may be more sensitive than the current standard for detecting early LD, while maintaining 100% specificity. We further show that analysis of relative antibody levels to predict disease status, such as in acute and convalescent stages of infection, is possible with a highly sensitive and quantitative platform like GC-FP. The results from this study add to the urgent conversation regarding better diagnostic strategies and more effective treatment for patients affected by tick-borne disease.