The modification of proteins in Mycobacterium tuberculosis (Mtb) by the prokaryotic ubiquitin-like protein (Pup) targets them for degradation by mycobacterial proteasomes. Although functionally similar to eukaryotic deubiquitylating enzymes, the deamidase of Pup (Dop) has no known mammalian homologs. Since Dop is necessary for persistent infection by Mtb, its selective inhibition holds potential for Tuberculosis therapy. In order to facilitate high-throughput screens for Dop inhibitors, we developed a time-resolved Förster resonance energy transfer (TR-FRET)-based assay for Dop function. The TR-FRET assay was successfully applied to determine the Michaelis constant for ATP-binding and to test the co-factor tolerance of Dop.