Active-site directed probes are a powerful tool to the study of enzymatic function. Here we report an active site directed probe based on a warhead so far considered unreactive. By replacing the C-terminal carboxylate of ubiquitin with an alkyne functionality, a selective reaction with the active site cysteine residue of deubiquitinating enzymes was observed. The resulting product was shown to be a quaternary vinyl thioether, as determined by X-ray crystallography. Proteomic analysis of proteins bound to an immobilized ubiquitin alkyne probe confirmed the selectivity towards deubiquitinating en-zymes. The observed reactivity is not just restricted to propargylated ubiquitin, as highlighted by the selective reaction between caspase-1 (Interleukin Converting Enzyme, ICE) and a propargylated peptide derived from IL-1?, a caspase-1 substrate.