Objective: Antimicrobial peptides (AMPs), as well as their antiparasitic analogues, are becoming increasingly important for combating hospital-acquired (hospital) infections. Proline- and tryptophan-rich AMPs (PR- and TR-AMPs) are resistant to classical trypsin proteinases, but can be cleaved by oligopeptidases B (OpdB), which participate in protecting bacteria against AMPs. The aim of this study was to investigate the proteolytic activity of OpdB from Serratia proteamaculans (PSP) against model substrates, which are an antiparasitic peptide containing an Arg-Pro peptide bond and its truncated fragment containing a Lys residue in the N-terminal position. Methods: The results of the PSP-dependent peptide hydrolysis were assessed using HPLC and MALDI-TOF mass spectrometry. Results and Discussion: It was found that PSP hydrolyzes the Arg-Pro peptide bond and has aminopeptidase activity. A method was developed for quantitative assessment of the efficiency of cleavage of the model peptides containing several hydrolysis sites. Conclusions: The obtained results confirm the significant role of OpdB in the defense system of bacterial and protozoan cells against PR- and TR-AMPs and emphasize the importance of searching for specific inhibitors of the enzyme.