Added PubMed Id The Toll-like receptor and chemotaxis pathways are key components of the innate immune system. Computational modeling and simulation at the molecular interaction level can be used to study complex biological pathways, but protein concentration values must be input as model parameters. In this investigation, targeted mass spectrometry assays were developed and used to measure the absolute abundance (copies/cell) of proteins of the mouse macrophage Toll-like receptor 4 (TLR4) and chemotaxis pathways. The data produced by this investigation can be used for pathway modeling and simulation, as well as for other systems biology research.