RING-Between-RING (RBR) E3 ligases mediate ubiquitin transfer through an obligate E3- ubiquitin thioester intermediately prior to substrate ubiquitination. While RBRs share a conserved catalytic module, substrate recruitment mechanisms remain enigmatic and the relevant domains have yet to be identified for any member of the class. Here we characterize the interaction between the auto-inhibited RBR, HHARI (AriH1), and its target protein, 4EHP, using quantitative XL-MS.