An increasing body of evidence indicates that constitutive activation of NF-kappaB contributes to tumorigenesis and inflammation. Ubiquitination and degradation of IkappaB plays an essential role in NF-kappaB activation. Here we describe an in vitro IkappaBalpha ubiquitination assay system in which purified E1, E2, SCF(beta-Trcp1) E3, IkappaBalpha, IKK2, and Ub were used to generate ubiquitinated IkappaBalpha. The ubiquitination of IkappaBalpha is strictly dependent on its phosphorylation by IKK2, as well as the presence of E1, E2, E3, and Ub. The assay was adapted into 384-well plate format in which an antibody against IkappaBalpha was used to capture IkappaBalpha, and the biotinylated ubiquitin attached to IkappaBalpha was detected with europium (Eu)-labeled streptavidin. This assay can be used to discover inhibitors of IkappaBalpha ubiquitination. Such inhibitors would block NF-kappaB activation by stabilizing IkappaB levels in cells and thus provide a new therapeutic approach to NF-kappaB-related human diseases.