Genetic code expansion has proven invaluable to the elucidation of functions of defined protein modifications through the site-specific incorporation of noncanonical amino acids. The use of nonhydrolyzable derivatives of post-translational modifications can greatly increase site stoichiometry and half-life. Investigating acetyllysine reader domain (bromodomain) interactions with acetylated nonhistone proteins is challenging due to the limited tools available and dynamic nature of this post-translational modification. Here, we demonstrate that bromodomains bind acetyllysine peptides and those substituted with an acetyllysine derivative, trifluoroacetyllysine, with similar affinity and selectivity. Importantly, both trifluoroacetyllysine and acetyllysine can be site-specifically incorporated into proteins expressed in bacterial and mammalian cells, and the strong electron-withdrawing trifluoro substituent makes the latter resistant to deacetylation by sirtuins (SIRTs). The controlled expression of SIRT-resistant, site-specifically acetylated transcription factors expands the set of available tools for determining the function of acetylation, and it serves as a template for investigating bromodomain interactions with acetylated transcription factors.