Covalent modifications of proteins with ubiquitin and ubiquitin-like molecules are instrumental to most, if not all biological processes. However, identifying the E3 ligase responsible for these modifications remains a major bottleneck in ubiquitin research. Here, we have developed an E2-thioester-driven identification (E2~dID) method for the targeted identification of substrates of specific E2 and E3 enzyme pairs. E2~dID exploits the central position of E2 conjugating enzymes in the ubiquitination cascade and provides in vitro generated biotinylated E2~ubiquitin thioester conjugates as the sole source for ubiquitination in extracto. This enables purification and identification of modified proteins by mass spectrometry under stringent conditions independently of the biological source of the extract. We demonstrate the sensitivity and specificity of E2-dID by identifying and validating substrates of the APC/C in human cells. Finally, performing E2~dID with SUMO in S. cerevisiae we show that E2-dID can be easily adapted to other ubiquitin-like modifiers and experimental models.