During antibiotic persistence, bacterial cells become transiently tolerant to antibiotics by restraining their growth and metabolic activity. Detailed molecular characterization of antibiotic persistence is hindered by low count of persisting cells and the need for their isolation. Here, we used sustained addition of stable isotopelabeled lysine to selectively label the proteome during hipA-induced persistence and hipB-induced resuscitation of Escherichia coli cells in minimal medium after antibiotic treatment. Time-resolved, 24-h measurement of label incorporation allowed detection of over 500 newly synthesized proteins in viable cells, demonstrating low but widespread protein synthesis during persistence. Many essential proteins were newly synthesized, and several ribosome-associated proteins such as RaiA and Sra showed high synthesis levels, pointing to their roles in maintenance of persistence. At the onset of resuscitation, cells synthesized the ribosome-splitting GTPase HflX and various ABC transporters, restored translation machinery, and resumed metabolism by inducing glycolysis and biosynthesis of amino acids. IMPORTANCE While bactericidal antibiotics typically require actively growing cells to exploit their function, persister cells are slowly replicating which makes them tolerant to the lethal action of antimicrobials. Here, we used an established in vitro model of bacterial persistence based on overexpression of the paradigm toxin-antitoxin (TA) system hipA/hipB to devise a generic method for temporal analysis of protein synthesis during toxin-induced persistence and antitoxin-mediated resuscitation. Our time-resolved, 24-h measurement of label incorporation demonstrated low but widespread protein synthesis during persistence. At the onset of resuscitation, cells restored translation machinery and resumed metabolism by inducing glycolysis and biosynthesis of amino acids. Our study provides the first global analysis of protein synthesis in persisting and resuscitating bacterial cells, and as such, presents an unprecedented resource to study the processes governing antibiotic persistence.