The capacity of buffalo milk proteins to release bioactive peptides was evaluated and novel bioactive peptides were identified. The sequential similarity between buffalo milk proteins and their cow counterparts was analysed. Buffalo milk proteins were simulated to yield theoretical peptides via in silico proteolysis. The potential of selected proteins to release specific bioactive peptides was evaluated by the A value obtained from the BIOPEP-UWM database (Minkiewicz et al. in Int J Mol Sci 20(23):5978, 2019). Buffalo milk protein is a suitable precursor to produce bioactive peptides, particularly dipeptidyl peptidase IV (DPP-IV) and angiotensin I-converting enzyme (ACE) inhibitory peptides. Two novel ACE inhibitory peptides (KPW and RGP) and four potential DPP-IV inhibitory peptides (RGP, KPW, FPK and KFTW) derived from in silico proteolysis of buffalo milk proteins were screened using different integrated bioinformatic approaches (PeptideRanker, Innovagen, peptide-cutter and molecular docking). The Lineweaver-Burk plots showed that KPW (IC = 136.28 ± 10.77 μM) and RGP (104.72 ± 8.37 μM) acted as a competitive inhibitor against ACE. Similarly, KFTW (IC = 873.92 ± 32.89 μM) was also a competitive inhibitor of DPP-IV, while KPW and FPK (82.52 ± 10.37 and 126.57 ± 8.45 μM, respectively) were mixed-type inhibitors. It should be emphasized that this study does not involve any clinical trial.