This probe report describes the identification and development of a potent, sub-micromolar (IC50 = 781 nM), first-in-class, small molecule inhibitor of Ubc13 enzyme activity, ML307. The activity of the probe compound was assessed by its ability to suppress Ubc13-mediated formation of heteropolymeric poly-ubquitination chains comprised of a mix of terbium-labeled and fluorescein-labeled ubiquitin molecules, as monitored by a reduction of the time-resolved fluorescent resonance energy transfer (TR-FRET) signal arising from this chains when synthesized in vitro in collaboration with the Ubc13 cofactor UEV1a. ML307 has excellent solubility and stability in buffer and is not a general cysteine protease inhibitor as it is >128-fold selective against Caspase-3, nor a TR-FRET artifact as it is not inhibitory a TR-FRET assay for Bfl-1, an out of class target. ML307 resulted from an extensive elucidation of the structure-activity relationship (SAR) through the purchase and synthesis of over 90 compounds representing 5 related scaffolds. The level of in vitro potency achieved is unprecedented in the current state of art, thus representing a significant advance and providing a chemical tool for assessing the biological roles and biochemical mechanisms of Ubc13.