Many protein-derived bioactive peptides were identified after extensive activity-guided purification, which is labor-intensive and costly. Furthermore, the rationale behind the selection of a substrate protein and a protease over others has not been justified in literature. The purpose of the study was to explore the rationale behind the selection of conditions for the production of potent angiotensin I converting enzyme (ACE) inhibitory peptides from egg proteins. Based on in silico digestion and quantitative structure and activity relationship (QSAR) model prediction, thermolysin-pepsin digestion of ovotransferrin was chosen as the best condition due to the presence of three potent peptides, Ile-Arg-Try, Leu-Lys-Pro and Ile-Gln-Try. To our surprise, sequences of Ile-Arg-Try-Cys-Thr, Leu-Lys-Pro-Ile and Ile-Gln-Try-Cys-Ala, but not Ile-Arg-Try, Leu-Lys-Pro and Ile-Gln-Try, were present in the hydrolysate. Further study showed that sonication or reducing agent pre-treatments could improve the activity of hydrolysates over 20 times and the predicted peptides were successfully released from sonication-treated ovotransferrin hydrolysate.