We report a comprehensive analysis of binding energy hot spots at the protein-protein interaction (PPI) interface between nuclear factor kappa B (NF-kappa B) essential modulator (NEMO) and I kappa B kinase subunit beta (IKK beta), an interaction that is critical for NF-kappa B pathway signaling, using experimental alanine scanning mutagenesis and also the FTMap method for computational fragment screening. The experimental results confirm that the previously identified NEMO binding domain (NBD) region of IKK beta contains the highest concentration of hot-spot residues, the strongest of which are W739, W741, and L742 (Delta Delta G = 4.3, 3.5, and 3.2 kcal/mol, respectively). The region occupied by these residues defines a potentially druggable binding site on NEMO that extends for similar to 16 angstrom to additionally include the regions that bind IKK beta L737 and F734. NBD residues D738 and S740 are also important for binding but do not make direct contact with NEMO, instead likely acting to stabilize the active conformation of surrounding residues. We additionally found two previously unknown hot-spot regions centered on IKK beta residues L708/V709 and L719/I723. The computational approach successfully identified all three hot-spot regions on IKK beta. Moreover, the method was able to accurately quantify the energetic importance of all hot-spot residues involving direct contact with NEMO. Our results provide new information to guide the discovery of small-molecule inhibitors that target the NEMO/IKK beta interaction. They additionally clarify the structural and energetic complementarity between "pocket-forming" and "pocket-occupying" hot-spot residues, and further validate computational fragment mapping as a method for identifying hot spots at PPI interfaces.