SDE2, a highly conserved and essential gene required for genome maintenance, coordinates essential processes ranging from pre-mRNA splicing and ribosome biogenesis to DNA repair and replication stress response. It is expressed as a full-length precursor (SDE2FL,) bearing an N-terminal ULD, which is proteolytically cleaved to generate two protein fragments, SDE2UBL and the larger C-terminal domain (SDE2CT). Here we report the use of activity-based probe to aid the identification of the protease responsible for SDE2 processing, resolving a previously uncharacterized mechanism of post-translational regulation.