Ubiquitination is a dynamic post-translational modification that regulates nearly all aspects of cellular physiology. Deubiquitinases (DUBs) remove ubiquitin from previously ubiquitinated substrates. Recent research indicates that ubiquitination plays a crucial role in extracellular vesicle (EV) biogenesis. Specific ubiquitin (Ub) linkages can lead to distinct functional outcomes. For example, K48 chains target proteins for degradation by the proteasome, while K63 chains function in endocytosis and lysosomal degradation. Our recent findings demonstrate that Arrdc4, an α-arrestin protein involved in EV secretion, is ubiquitinated by the Nedd4-2 E3 ubiquitin ligase at lysine 270 (K270) with Ub K29 chains. This specific ubiquitination is critical for Arrdc4’s role in EV secretion. To further elucidate the molecular mechanisms of Arrdc4 K29-dependent EV biogenesis, we utilised TRABID, a DUB that specifically cleaves Ub K29 and K33 chains. We discovered that TRABID interacts with Arrdc4 and removes Ub K29 chains at K270, thus impeding Arrdc4-dependent EV release. Interestingly, overexpression of TRABID reduced overall release of EVs, while TRABID ablation increased EV secretion. These results demonstrate that Ub K29 linkage plays a regulatory role in EV biogenesis. Immunoprecipitation-mass spectrometry using a Ub K29-specific antibody identified over 500 Ub K29-modified proteins, with subsequent bioinformatics analysis suggesting that some Ub K29-modified proteins may participate in EV biogenesis regulation. We have characterised one such candidate with a previously known function in EV biology that is modified with Ub K29 chains and is a substrate for TRABID. We also identified an E3 ligase likely responsible for the K29 ubiquitination. We are currently investigating how these regulatory networks control EV biogenesis.