The NlpC/p60-family of peptidoglycan hydrolases are key enzymes that facilitate bacterial cell division and also modulate microbe-host interactions. These endopeptidases utilize conserved Cys-His residues in their active site and are expressed in most bacterial species as well as some eukaryotes. Here we describe methods for biochemical analysis of Enterococcus faecium SagA-NlpC/p60 peptidoglycan hydrolase activity (Kim et al., 2019; Rangan et al., 2016), which includes recombinant protein preparation and biochemical analysis using both gel-based and LC-MS profiling of peptidoglycan fragments. These protocols should also facilitate the biochemical analysis of other NlpC/p60 peptidoglycan hydrolases.