The use of noncanonical amino acids (ncAAs) to control the viability of an organism provides a strategy for the development of conditional “kill switches” for live vaccines or engineered human cells. We report an approach inspired by the posttranslational acetylation/deacetylation of lysine residues, in which a protein encoded by a gene with an in‐frame nonsense codon at an essential lysine can be expressed in its native state only upon genetic incorporation of N‐ϵ‐acetyl‐l‐Lys (AcK), and subsequent enzymatic deacetylation in the host cell. We applied this strategy to two essential E. coli enzymes: the branched‐chain aminotransferase BCAT and the DNA replication initiator protein DnaA. We also devised a barnase‐based conditional suicide switch to further lower the escape frequency of the host cells. This strategy offers a number of attractive features for controlling host viability, including a single small‐molecule‐based kill switch, low escape frequency, and unaffected protein function.