Cysteines residues are poorly conserved across protein classes, and represent one of the least abundant natural amino acids (2% of total residues). Their nucleophilicity enables reaction with reactive partners including acrylamides, haloacetamides, disulfides, and α,β-unsaturated esters. Inclusion of these moieties into the protein ligand results in formation of a covalent bond with the cysteine-containing protein giving a prolonged residence time, a concomitant increase in affinity, potency, and often selectivity. This approach can be employed for WT proteins that contain a poorly conserved cysteine, and potentially for engineered proteins that include a mutant cysteine residue. One of the pioneer studies exploiting cysteine chemistry is the tethering approach, in which the presence of native or engineered cysteines is capitalized upon to discover new small molecule inhibitors via the formation of a disulfide bond (Figure ). The aim of our work was to develop a technology that combines site-directed cysteine mutation with covalent fragments allowing rapid elucidation of ligands to inhibit the function of the mutant protein domain (Figure ). The advantage of this approach is that the covalent nature of the ligand avoids the need for iterative rounds of development to increase affinity for the target protein. Additionally, the combination of the mutant cysteine and the electrophilic ligand can provide selectivity over other possible off-targets. A similar approach to the development of a selective ligand for the EphB1 kinase was reported by Kung et al., but this work employed an already established kinase ligand, rather than electrophilic fragments.