The objective of this study was to investigate the influence of the concentration of Al3+ ions and the substrate/enzyme ratio on pepsin and trypsin activity in vitro. The experimental design was a combination of three Al3+ ion concentrations (0.25; 2.5 and 25.0 mg Al3+/ml of reaction solution) and two substrate/enzyme ratios (S/E = 10 and 100 for pepsin, and 100 and 1000 for trypsin). Enzymatic activity was determined by the Folin method based on the reaction of tyrosine with the Folin reagent. It was found that the concentration of Al3+ ions influenced activity of pepsin which increased with the increasing metal ion concentration in the reaction milieu. Al3+ ions did not affect activity of trypsin. Proteolitic activity of pepsin and trypsin depended on substrate/enzyme ratio. Higher concentration of substrate decreased efficacy of enzymatic protein breakdown in vitro.