Proteins targeted for degradation by the mycobacterial proteasome are covalently modified with prokaryotic ubiquitin-like protein (Pup) in a process termed "pupylation." Despite its name, Pup is only ubiquitin-like in function and not sequence or structure. Furthermore, the enzymology of pupylation appears to be distinct from protein modification by ubiquitin (Ub) and other ubiquitin-like proteins (Ubls). Nonetheless, we have adapted methods established in the Ub field for the production of reagents to isolate, identify, and analyze pupylated proteins in mycobacteria. These methods can be modified to study specific pupylated proteins in various Pup-bearing bacteria or to identify posttranslational modifiers in other prokaryotes.