The innate immune system is the first line of defense against pathogens. Pattern recognition receptors (PRRs), such as the Toll-like receptors (TLRs) sense and sample pathogen-associated molecular patterns (PAMPs). On the host myeloid cell surface, the proinflammatory Gram-negative bacterial outer membrane component lipopolysaccharide (LPS, also known as endotoxin) activates the innate immune system via TLR4. Intracellularly, LPS is detected by the noncanonical inflammasome through caspase4/5/11. In the present work, mass spectrometry (MS)-based top-down structural analysis of LPS uncovered major determinants of molecular pathogenesis, and MS-based systems immunoproteomics elucidated specific features of the immune response against endotoxin. We used targeted proteomics to profile the host response to the pathogens Escherichia coli, Staphylococcus aureus, and Burkholderia cenocepacia, and we discovered significant temporal changes in the macrophage secretome. Additionally, we identified global changes in protein secretion in TLR4- and caspase11- stimulated macrophages. Finally, we observed bacterial proteomic rewiring within the biofilm forms of Burkholderia, possibly explaining the observed lowering in sensitivity to antibiotics.