Synthetic heptapeptide containing D-amino acid residues (Val-D-Leu-Pro-Phe-Phe-Val-D-Leu) was coupled to glyoxal-activated magnetic agarose particles via the free peptide amino group. The peptide-modified magnetic particles were used for the separation of pepsins. Porcine pepsin A and human pepsin A were adsorbed to the magnetic peptide-modified affinity carrier, while the rat pepsin C and human pepsin C did not interact with the immobilized ligand. Conditions of pepsin adsorption to peptide-modified magnetic particles, as well as elution buffers were optimized. Porcine pepsin A did not interact with the immobilized peptide in the presence of pepsin inhibitor pepstatin A, indicating that the enzyme binding site is involved in the studied interaction. The elaborated method represents a rapid and simple technique not only for the separation of pepsins but also, in combination with MS, for the enzyme detection and determination.