KB05yne is a site-selective, alkyne-functionalized electrophilic "fragment" probe, crafted for covalent ligand discovery with preferential reactivity towards specific cysteine residues in proteins. It robustly labels wild-type target proteins (such as CDKN2AIP, HPCAL1) but not their cysteine-to-alanine mutants, confirming site-specific cysteine reactivity. KB05yne is utilized in the discovery and optimization of site-specific covalent ligands for a range of structurally and functionally diverse proteins, particularly in studying cysteine residues within cancer-related or hard-to-purify proteins.