The ubiquitin (Ub) system regulates a wide range of cellular signaling pathways. Several hundred E1, E2, and E3 enzymes are together responsible for protein ubiquitination, thereby controlling cellular activities. Due to the numerous enzymes and processes involved, studies of ubiquitination activities have been challenging. We here report a novel Förster resonance energy transfer (FRET)-based assay for studying the in vitro kinetics of ubiquitination. FRET is established upon binding of fluorophore-labeled Ub to eGFP-tagged ZnUBP, a domain that exclusively binds unconjugated Ub. We name this assay the free Ub sensor system (FUSS). Using Uba1, UbcH5, and CHIP as model E1, E2, and E3 enzymes, respectively, we demonstrate that ubiquitination results in decreasing FRET efficiency, from which reaction rates can be determined. Further treatment with USP21, a deubiquitinase, leads to increased FRET efficiency, confirming the reversibility of the assay. We subsequently use this assay to show that increasing the concentration of CHIP or UbcH5 but not Uba1 enhances ubiquitination rates and develop a novel machine learning approach to model ubiquitination. The overall ubiquitination activity is also increased upon incubation with tau, a substrate of CHIP. Our data together demonstrate the versatile applications of a novel ubiquitination assay that does not require labeling of E1, E2, E3, or substrates and is thus likely compatible with any E1-E2-E3 combinations.