The M16A peptidase subfamily comprises a group of zinc-dependent enzymes that are highly conserved among both prokaryotic and eukaryotic organisms. In this study, we have developed and used multiple new tools to better characterize the biochemical and physiological properties of prokaryotic and eukaryotic M16A peptidases. We have evaluated structure/function relationships of M16A peptidases in the context of Eschericia coli pitrilysin, the first M16A enzyme for which structural data became available, investigated the conserved enzymatic properties of the M16A subfamily in the context of Saccharomyces cerevisiae Ste23p, the most representative yeast M16A homolog, and developed capillary electrophoresis as a viable and efficient technique for evaluating M16A proteolysis in vitro, particularly within the context of mammalian insulin-degrading enzyme mediated cleavage of the amyloid beta 1-40 peptide.