LAUSR

Activity‐based probes (ABPs) are valuable chemical tools for profiling enzymes. They have been particularly useful in the study of proteases. ABPs rely on electrophilic scaffolds that covalently modify the target enzymes. Ideally, they can be made in a fast and uncomplicated manner. Here, we explore alkyne‐substituted benzoxazin‐4‐ones as ABPs for serine proteases, because they inhibitserine proteases covalently and their synthesis is very straightforward. We show that alkyne‐tagged benzoxazin‐4‐ones can be used in two‐step bioorthogonal tandem labeling procedures or pre‐functionalized with a biotin or fluorophore. We demonstrate that these reagents can be used to label and identify various serine proteases. Therefore, we expect that tagged benzoxazin‐4‐ones will offer easily synthesizable tools for profiling of serine proteases.