Protein posttranslational reversible lysine N^ε-acetylation and deacetylation have been recognized as an emerging intracellular signaling mechanism that plays critical roles in regulating gene transcription, cell-cycle progression, apoptosis, DNA repair, and cytoskeletal organization. Acetyltransferase-catalyzed creation, deacetylase-catalyzed destruction, and bromodomain-mediated specific recognition of N^ε-acetyl-lysine on proteins define the central events of this signaling mechanism. In our efforts toward developing novel inhibitors of the bromodomain/N^ε-acetyl-lysine recognition, whose chemical modulating strategies and modulators are still under-developed as compared to other events involved in this signaling mechanism, we developed the first non-hydrolyzable (or intracellular protein deacetylase-resistant) functional surrogate, i.e. N^ε-methanesulfonyl-lysine, for N^ε-acetyl-lysine regarding bromodomain binding interaction. In specific, our experimental results suggested that N^ε-methanesulfonyl-lysine replacement for N^ε-acetyl-lysine i) did not compromise the binding affinity for the bromodomain, ii) conferred resistance to protein deacetylases, and iii) conferred only weak inhibition against protein deacetylases. The availability of this non-hydrolyzable analog will not only promote the inhibitor development, but also facilitate the functional examination of protein posttranslational acetylation due to its capability to provide the constitutive phenotype of protein acetylation.