We investigated the effects of extracellular proteinases of two Pseudomonas aeruginosa clinical isolates on the essential humoral immune response parameters in hemolymph of the insect model organism Galleria mellonella in vitro. Two culture media, rich LB and minimal M9, known to induce synthesis of different sets of proteinases secreted by P. aeruginosa were used. Changes in lysozyme, antibacterial and antifungal activities, as well as protein and peptide patterns in hemolymph treated with proteolytic fractions were evaluated. The effect of the proteolytic fractions on the apoLp-III level in hemolymph was determined by immunoblotting with antibodies against G. mellonella apolipophorin III (apoLp-III). We found that apoLp-III is hardly degraded by the proteinases of the proteolytic fractions of both clinical P. aeruginosa strains, in contrast to the high susceptibility of the protein to the proteinases of the entomopathogenic strain. The detected differences, together with the changes in the hemolymph protein and peptide patterns caused by the studied fractions, reflected the distinct composition of secreted proteinases of the entomopathogenic P. aeruginosa strain and the clinical strains tested. Our results also suggest the involvement of alkaline protease, the main proteinase of proteolytic fractions of P. aeruginosa grown in minimal medium, in the degradation of G. mellonella antimicrobial factors, such as lysozyme, antibacterial polypeptides, and proteins with antifungal activity. The diverse effects of the P. aeruginosa proteolytic fractions studied on the parameters of G. mellonella immune response indicate that this model insect may be useful in the analysis of the virulence factors of different P. aeruginosa strains.