Our homogeneous Real-Rime TR-FRET ubiquitin conjugation assays are simple; the format is 96 or 384 well low-volume plates, and assays are 'set and forget'. A mixture of ubiquitins labeled with cryptate and acceptor fluorophore are combined with UBA1 activating enzyme, E2 conjugating enzyme, E3 ligase, substrate protein (optional), 10x reaction buffer, and initiated with addition of ATP. As donor and acceptor ubiquitins become incorporated into chains they come into close proximity, resulting in energy transfer (FRET) and signal detection. Readout can be collected in Real-Time, facilitating enzyme kinetics, or endpoint if preferred. E3 ligase may be used in the reaction to target ubiquitination of ligase substrates, but care must be taken to avoid concurrent ligase auto-ubiquitination. Assay performance is typically unparalleled; Z’ are usually >0.8, Signal to Noise is commonly > 3000%.