Abstract During the past 30 years, notable advances have been made in understanding the etiologic agent, Borrelia burgdorferi (Bb), and the illness that it causes. While ~30,000 cases are reported to the CDC each year, the overall number of Bb-infected patients in the US is estimated to approach 400,000, suggesting that Lyme disease is becoming an epidemic. Signs and symptoms of infection range in severity and most patients recover fully after antimicrobial treatment; however, chronic serious illness and even deaths can still occur. Lyme disease is usually diagnosed by clinical observation of erythema migrans, however, some patients do not develop erythema migrans. Why certain patients have no accompanying symptoms at time of presentation whereas others have numerous symptoms has not been elucidated. Many studies have been published detailing progress in expanding the knowledge base on the microbiology of Bb and on the ecology and epidemiology, pathogenesis and clinical aspects but laboratory based diagnosis of Lyme disease is severely lagging behind. It is been estimated that more than 2.7 million serum samples are tested each year for the presence of Borrelia burgdorferi-specific antibodies in the United States alone. To meet the demand for laboratory-based diagnosis, various new tests for direct detection of the etiologic agent, or for detection of post-infection specific antibodies by using whole-cell lysates, recombinant antigens, or peptide antigens in enzyme immunoassays (EIA), have been introduced into the clinical laboratory. However, the currently available Lyme disease diagnostics do not meet the specifications for an ideal test, which would be rapid, sensitive, specific, and point-of-care. Currently, the two step FDA approved diagnostic test will only detect the post-infection immune response to Lyme disease pathogen, Bb, but with limited sensitivity and specificity. An ideal Lyme disease diagnostic is a test that is specific for Bb, simple, non-invasive and relies only on readily available samples such as blood or urine. Our proposal will provide specific targets detectable in blood at the earliest time point upon Bb infection. To develop these tests, we will develop a comprehensive quantitative pathogen surfaceome and targeted quantitative Bb protein remnant detection in complex host proteome backgrounds for early Lyme disease detection. We will organize all our data into a publically accessible ?Borrelia PeptideAtlas and Borrelia SRMAtlas? to provide an ongoing resource for Lyme researchers. This proposal will have great impact and contribution to reduce disproportionate identification of Lyme disease through the establishment of novel biomarkers that can stratify patients. The outcome of this project is the identification and verification of highly specific and highly sensitive pathogen targets for development into simple diagnostic assays for early stage Lyme disease patients.