The degree of hydrolysis, as measured by potentiometric titration in the presence of formaldehyde, and free radical scavenging rate in five bioenzymes, namely flavor proteases, alkaline proteases, papains, neutral proteases, and pepsins were compared to identify the bioenzyme with the best proteolytic performance against the Prunus dulcis var. amara prolamins under optimal conditions. The effects of independent variables such as enzyme addition, substrate concentration, pH and reaction time were explored via single-factor experiments. Box-Behnken response surface methodology considering these four factors at three levels was then used to optimize the proteolysis of these five bioenzymes. The results demonstrate that alkaline proteases outperform the other enzymes when using the Prunus dulcis var. amara prolamins as substrate. The optimal conditions were determined to be as follows: a reaction time of 4.12 h; a total of 5208.93 U/g of the enzyme should be added; the substrate concentration should not exceed 3.21%; and the pH should be maintained at 9.22. Final evaluations using these conditions allowed for an additional round of refinement and we determined that the optimal conditions are as follows: a reaction time of 4.0 h; 5000.0 U/g of enzyme; substrate at 3.0%; and the pH at 9.0. These conditions were then validated by the evaluation of the end product with these processed petides increasing the DH% and DPPH free radical scavenging rate to 26.74%±0.54% and 97.86%±0.58%, respectively. These values are very similar to the predicted values from our in silico evaluation, confirming that these are the optimal conditions for the proteolysis of the prolamin proteins produced by Prunus dulcis var. amara.