The Urm1 protein, a member of the ubiquitin (Ub)-related modifier family, is highly conserved across evolution and plays a crucial role as a sulfur donor in tRNA thiolation. Despite its importance, Urm1 remains relatively understudied in mammalian eukaryotes, leading to numerous unknown functions. As of now, no deubiquitinases that interact with Urm1 in mammalian cells have been reported, nor are there known conjugating enzymes (E2), or ligases (E3) involved in the Urm1 cascade. Our study introduces an activity-based Urm1 probe featuring a C-terminal alkyne group, which specifically targets the active-site cysteine residues of deubiquitinases and other key enzymes in the degradation pathway. Using a proteomic approach, we identified deubiquitinases, E1, and E2 enzyme that bind with Urm1 in mammalian cells, paving the way for further exploration and understanding of Urm1's functional interactions.