Interleukin 1 receptor activation innervates a cascade of signal transduction events that ultimately lead to the activation of inflammatory and immune response genes. TRAF6 is a Ub ligase (E3) involved in this pathway, and inhibition of this critical enzyme may provide a means for treating inflammatory and immune diseases. A TR-FRET assay has been developed and evaluated for HTS for TRAF6 inhibitors. Bio-Ub and Eu-Ub were polymerized in the presence of Ub activating enzyme E1, conjugating enzyme E2, and TRAF6. Following a 2-h incubation, the reaction was stopped with a buffer containing 10 m M EDTA and the fluorescence donor SA-APC. Fluorescence energy transfer from Eu to APC was measured as a ratio of fluorescence intensity at 655 nm to that at 615 nm (excitation at 340 nm). This homogeneous assay has been optimized and validated in a 384-well format. A window of five- to eightfold and Z' factor of 0.6-0.8 suggests that this assay can be applied to screen for inhibitors of the polyubiquitination activity of TRAF6.