The N-end rule pathway has emerged as a major system for controlling protein stability in medical, animal and plant sciences as well as agriculture. Although, novel functions and enzymes of the pathway were discovered, ubiquitination mechanism and substrate specificity of N-end rule pathway E3 Ubiquitin ligases remained elusive. Taking the first discovered bone fide plant N-end rule E3 ligase PROTEOLYSIS1 (PRT1) as a model, we describe a novel tool to molecularly characterize polyubiquitination live, in real-time. We demonstrated for the first time that PRT1 is indeed an E3 ligase, which was hypothesized for a long time. We gained mechanistic insights in PRT1 substrate preference and activation by monitoring live ubiquitination by using a fluorescent chemical probe coupled to artificial substrate reporters. Ubiquitination can then be measured by rapid in-gel fluorescence scanning in classical end-point assays as well as in real time by fluorescence polarization in standard microplate readers. Enzymatic activity, substrate specificity, reaction mechanisms and optimization can be easily investigated ad hoc in short time and with significantly reduced reagent consumption. These results demonstrate that PRT1 has the potential to be involved in polyubiquitination of various substrates and therefore pave the way to understand recently discovered phenotypes of prt1 mutants.