Computational biochemistry methods (molecular docking and molecular dynamics) had become present-day and timely approaches for theoretical research of interaction between hyaluronidase and glycosaminoglycans (GAG). Stabilizing effect of chondroitin ligands on thermal inactivation of enzyme was demonstrated. This effect was followed by reinforcement of molecular structure of biocatalyst, increase of thermal denaturation temperature by 10—15 °C and inhibiting the blockade of enzyme active site entrance. Molecular docking with its subsequent elaboration by molecular dynamics method is a productive approach for research of protein-glycosaminoglycan interaction. Competitive reaction of chondroitin sulfate trimers and heparin tetramers with hyaluronidase contributes to enzyme endoglycosidase activity regulation altering its heparin-mediated inhibition. The sequence of preferable GAG ligand binding determined in our study has underlied the guidelines for practical regulation of hyaluronidase activity via its experimental chemical modification. Modification with chondroitin sulfate trimers against cs7 or cs7, cs1 and cs5 centers on molecular surface of biocatalyst theoretically protects the enzyme from heparin-mediated inhibition. Determination of optimal GAG regulators of endoglycosidase activity of hyaluronidase is based on systematic theoretical research of limiting enzyme activity interactions with microenvironment components of biocatalyst with their subsequent experimental verification.