Updated publication reference for DOI(s): 10.1101/763029. Amblyomma americanum ticks transmit more than a third of human tick-borne disease (TBD) agents in the United States. Tick saliva proteins are critical to success of ticks as vectors of TBD agents, and thus might serve as targets in tick antigen-based vaccines to prevent TBD infections. We describe a systems biology approach to identify, by LC-MS/MS, saliva proteins (tick=1182, rabbit=335) that A. americanum ticks likely inject into the host every 24 h during the first 8 days of feeding, and towards the end of feeding using two different sample preparation approaches (in-gel and in-solution). While the in-gel approach provided a link between the protein size and identification, the in-solution approach provided more depth in identifying an extensive list of proteins. Putative annotation grouped tick and rabbit proteins in A. americanum tick saliva into 27 and 25 functional categories, respectively. Similar to Prostriata Ixodes scapularis ticks, the most predominant functional category of proteins in A. americanum is tick-specific (32%). Other notable abundant saliva protein categories include protease inhibitors (13%), proteases (8%), glycine-rich proteins (6%) and lipocalins (4%). This study reveals several biologically relevant protein categories present in tick saliva that are important to tick feeding physiology and regulation of tick and host interactions, and highlights the potential role(s) of these proteins in evading host defense mechanisms. Of significance, 284 A. americanum tick saliva proteins are also secreted in saliva of other ticks and might represent those that regulate key tick feeding functions. We discuss our findings in the context of A. americanum tick feeding physiology.