In this work the efficient and simple method of improvement specificity and solubility of low molecular weight proteinase substrates is described. The series of fluorescent substrates of selected proteolytic enzymes (neutrophil elastase, cathepsin G and proteinase 3 along with human airway trypsin like protease) were synthesized and modified by selective pegylation by the attachment of 2-(2-(2-aminoethoxy)ethoxy)acetic acid. Modification of the C-terminal carboxyl group resulted in the decrease in the specificity constants (kcat/KM) for all obtained analogues. The covalent attachment of PEG to N-terminal amino group has the opposite effect, as the increase in specificity constant was observed for all studied compounds. This outcome was pronounced the most for proteinase 3 substrate PEG-ABZ-Tyr-Tyr-Abu-ANB-NH2, whose catalytic constant (kcat) increased over three fold. The introduction of PEG moieties at both C- and N-terminal yielded the substrates with lower specificity constants. For substrate (ABZ-Arg-Gln-Asp-Arg-ANB-NH2) the influence of the PEG chain length on its kinetic parameters was investigated. Elongation of the PEG chain at N-terminal of this peptide decreased the specificity constant. In addition to the effect of pegylation on the kinetic parameters of the studied substrates, the introduced modifications significantly improved their solubility in buffer solutions applied for enzymatic investigations.