XL1 are the most active components of the antimicrobial lysoamidase complex. A sandwich enzyme immunoassay based on monoclonal antibodies was developed for the quantitation of these enzymes in complex protein mixtures. Non-cross-reactive monoclonal antibodies were produced using various methods of immunization with mature protein forms and synthetic peptide conjugates. A total of 15 monoclonal antibodies to AlpA, obtained after immunization with native protein, recognized the conformational epitopes, whereas six monoclonal antibodies to AlpB recognized the denatured form of the endopeptidase, and were obtained after immunization with peptide conjugates, whose amino acid sequences corresponded to AlpB mature form primary structure fragments different from AlpA. The developed method enables a highly sensitive assay of homologous proteases AlpA and AlpB with the detection ranges of 0.2–3.1 and 3–100 ng/ml for the native form of AlpA and the denatured form of AlpB at a 5% measurement error, respectively. The technique can be used to assess the production of AlpA and AlpB, quantitate these proteins in lysoamidase and study their secretion into the environment.