Nuclear factor-κB (NF-κB) is an inducible cytoplasmic transcription factor that plays a role as a master regulator of airway mucosal inflammation. The prototypical (“canonical”) NF-κB pathway controls cytoplasmic to nuclear translocation in response to stimulation by the mononuclear cytokine, TNF. Despite intensive investigation, the spectrum of kinases involved in the canonical NF-κB pathway has not yet been systematically determined. Here we have applied a high throughput siRNA-mediated loss-of-function screening assay to identify novel kinases important in TNF-induced NF-κB signaling. Type II A549 epithelial cells stably expressing an IL-8/luciferase reporter gene optimized for high throughput siRNA format (Z′ score of 0.65) and siRNAs for 636 human kinases were reverse-transfected and screened in the assay. 36 candidate genes were identified that inhibited TNF signaling with a Z score deviation of