Nuclear factor kappa B (NF-κB) is an inducible transcription factor that regulates the expression of many genes involved in normal immune and inflammatory responses. NF-κB activation is normally a rapid and transient response to pro-inflammatory stimuli however dysregulated constitutively active NF-κB signaling leads to chronic inflammation and provides a cell survival signal in many types of cancer. NF-κB signaling is therefore an important target for the development of novel anti-inflammatory or anti-cancer drugs. We previously identified and characterized a cell-permeable peptide that blocks NF-κB signaling by disrupting the critical upstream IκB kinase (IKK) complex. We describe in this chapter three separate methods to determine the effects of this NEMO-binding domain (NBD) peptide on pro-inflammatory NF-κB signaling in response to tumor necrosis factor (TNF).