The S‐alkylation of Cys residues with a maleimide and the Nϵ‐acylation of Lys residues with an N‐hydroxysuccinimide (NHS) ester are common methods for bioconjugation. Using Cys and Lys derivatives as proxies, we assessed differences in reactivity depending on the position of Cys or Lys in a protein sequence. We find that Cys position is exploitable to improve site‐selectivity in maleimide‐based modifications. Reactivity decreases substantially in the order N‐terminal>in‐chain>C‐terminal Cys due to modulation of sulfhydryl pKa by the α‐ammonium and carboxylate groups at the termini. A lower pKa value yields a larger fraction thiolate, which promotes selectivity while somewhat decreasing thiolate nucleophilicity in accord with βnormalnnormalunormalc =0.41. Lowering pH and salt concentration enhances selectivity still further. In contrast, differences in the reactivity of Lys towards an NHS ester were modest due to an appreciable decrease in amino group nucleophilicity with a lower pKa of its conjugate acid. Hence, site‐selective Lys modification protocols will require electrophiles other than NHS esters.