The transcription factor NF-κB is a key regulator of cellular activation, proliferation and apoptosis. Defects in the NF-κB pathway contribute to a broad array of malignant, neurodegenerative and chronic inflammatory diseases. IKK-dependent IκBα degradation by the 26S proteasome is a critical NF-κB regulatory control point, which is emerging as an important target for drug development. To directly monitor regulation of IKK activation in intact organisms, we engineered an IκBα–firefly luciferase (IκBα-FLuc) fusion reporter. In cultured cells and living animals, the reporter provided a continuous, noninvasive readout of the kinetics of ligand-induced IKK activation and the pharmacodynamics of selective inhibitors of both IKK and the 26S proteasome. This IκBα-FLuc reporter now permits continuous readout of IKK activation in vivo, facilitates development and validation of target-specific therapeutics, and complements conventional NF-κB transcriptional reporters for more complete temporal and regional investigations of the NF-κB signaling pathway in health and disease.