Understanding the molecular basis of how the tick adapts to feed on different animal hosts is central to understanding tick and tick-borne disease (TBD) epidemiology. Tick adaptation to feed on vertebrate hosts is regulated by tick secretion of multiple tick saliva proteins (TSPs) and other molecules that regulate tick feeding. This study was initiated to determine if ticks such as Ixodes scapularis and Amblyomma americanum that are adapted to feed on multiple hosts utilized the same sets of proteins to accomplish feeding on all hosts. Our data suggest that ticks of the same species differentially express proteins when feeding on diffent hosts. SDS-PAGE and silver staining analysis revealed unique protein eletrophoretic profile in saliva of Ixodes scapularis and Amblyomma americanum that were stimulated to start feeding on different hosts: rabbits, humans, and dogs. LC-MS/MS sequencing and pairwise analysis of proteins in saliva of I. scapularis and A. americanum ticks that were non-stimulated and those that were stimulated to feed on rabbits, dogs, or humans identified TSPs that were unique to each treatment and those that were common. Overal, we identified a total of 276 and 340 non-redundant I. scapularis and A. americanum TSPs, which we have classified into 28 functional classes that include secreted conserved proteins (unknown functions), proteinase inhibitors, lipocalins, extracellular matrix/cell adhesion, heme/iron metabolism, signal transduction and immunity-related proteins being the most predominant in saliva of unfed ticks. With exception of Rhipicephalus microplus, anti-tick vaccine research relies on feeding lab animals. Data here suggest that lab animal data could result in prioritizing irrelevant targets as some tick genes are unique to ticks fed on lab animals. This study provides the platform that could be utilized to identify relevant target anti-tick vaccine antigens, and will facilitate early stage tick feeding research.