Lyme disease (LD) is one of the most important human vector-borne diseases in North America. Since transmission of Borrelia burgdorferi (Bb), the causative agent of LD, is influenced by yet unknown tick saliva proteins (TSP), the discovery and characterization of such TSPs are highly sought after for their potential as tick-antigen based vaccine targets. We validated a novel non-invasive approach of collecting sufficient amounts of saliva from unfed, partially fed (12, 24, 36, 48, 60, and 72h), and replete fed Ixodes scapularis nymphs for identification of tick saliva proteins using the LC-MS/MS approach. Our data has described TSPs that might be injected into the host within few hours of the tick starting to feed and are likely associated with manipulating host immunity to facilitate transmitted Bb to colonize the host. Thus, these data will serve as a source for Bb-infection associated TSPs that might be targeted in tick-antigen based vaccines to prevent LD. Equally important, the non-invasive method to collect nymph tick saliva is likely to provide transformative impact on research to understand transmission of other TBD agents.