The innate immune system senses invariant microbial components via toll-like receptors (TLRs) to elicit a host defense program against invading pathogens. Lipopolysaccharide (LPS), a constituent of Gram-negative bacteria, is recognized by TLR4 and triggers protein kinase signaling to orchestrate immune responses such as inflammatory cytokine production. To analyze kinase-proximal signaling in murine macrophages, we performed prefractionation experiments with immobilized kinase inhibitors to enrich for protein kinases and their interaction partners. In conjunction with SILAC-based quantitative mass spectrometry and phosphopeptide enrichment, we recorded five time point profiles for more than 850 distinct phosphorylation events on protein kinases and copurifying factors. More than 15% exhibited significant changes and many of those mapped to LPS-regulated kinase networks. We identified many unreported TLR signaling events including LPS-triggered phosphorylations of Akt substrates, which point to previously unknown molecular mechanisms in innate immune response. We further detected extensive phosphoregulation of TANK-binding kinase 1, inhibitor of nuclear factor-kappaB kinase epsilon and their associating scaffolding factors, and none of these events were known despite the key roles of these proteins in LPS signaling. Thus, our data expands previous knowledge for functional analyses of innate immune response.