Peptide-based chemicals are promising for numerous applications including home and personal care and medical treatments. To better understand and control the environmental fate of peptide-based chemicals, in-depth knowledge on the specificity of wastewater peptidases is needed. Here, we employed multiplex substrate profiling by mass spectrometry to obtain specificity profiles of extracellular peptidases derived from influent and aeration tanks of three full-scale wastewater treatment plants. Specificities were confirmed by fluorogenic peptidase substrates. Our results revealed highly similar specificity profiles across wastewater treatment plants. We found that hydrolysis by extracellular wastewater peptidases is favored when positively charged amino acid residues surround the cleavage site and disfavored when negatively charged amino acid residues surround the cleavage site.