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Covalent inhibitors have numerous applications as drugs, as tools for drug discovery, and as probes for chemical biology. This class of compounds depends on the availability of a suitably reactive nucleophile in the target protein of interest. Among the non-catalytic nucleophiles, cysteine is the most reactive at physiological pH, but also the least prevalent. Based on the analysis presented herein, ~80% of known binding sites in the human proteome lack a cysteine residue. In contrast, ~80% of known binding sites contain either a lysine or a tyrosine—nucleophilic residues that are much less reactive than cysteine at physiological pH. Novel methods of covalent inhibition are therefore necessary to target a larger proportion of the proteome. We mined the Protein Data Bank (PDB) for small molecules within 5 Å of a nucleophilic atom (the epsilon amine in lysine, the phenol hydroxyl in tyrosine or the side chain thiol in cysteine). From the resulting compendium of proteins and ligands, we started with PU-H71 as an affinity scaffold to develop arylsulfonyl fluoride-based inhibitors that target a non-catalytic, non-reactive, surface-exposed lysine residue adjacent to the ATP binding site of Hsp90. We used high-resolution X-ray crystallography to decipher the molecular basis by which constrained, chiral linkers enantioselectively increase the reactivity of sulfonyl fluoride probes toward Hsp90 Lys58, without affecting the reversible binding affinity or intrinsic chemical reactivity. The in vivo use of covalent inhibitors requires electrophiles with the proper balance of metabolic stability and reactivity for their targets. We used the salicylaldehyde group as a reversible covalent electrophile to develop probes that react with the catalytic lysine in kinases. Using these probes, in combination with mass spectrometry, we found that ~50% of the kinome can react with the probes under equilibrium labeling conditions, including 80 kinases from living mice treated with the probes. Despite this promiscuity, we discovered that these probes exhibit remarkable kinetic selectivity for a small subset of kinases. The salicylaldehyde probes exhibit an apparent dissociation half-life of greater than 6 hours from a handful of kinases. These are among the first lysine-reactive kinase probes to exhibit the requisite stability and reactivity for in vivo applications. These novel probes desmonstrate how to achieve kinase selectivity, despite targeting the conserved, catalytically essential lysine residue.