Soybean protease hydrolysate (SPH) contains abundant antihypertensive peptides, yet the sequence defining vital antihypertensive peptides is unclear. Peptiomics sequencing analyzed peptides in enzymatic products from soybean protein alkaline and flavor proteases. Database queries and simulations screened key peptide monomers, assessing angiotensin-converting enzyme (ACE) inhibitory activities in vitro. Peptidomics identified 6,980 short peptides in SPH, predominantly hydrophobic at the N-terminal. Sixty-eight were target peptides, assessed for allergenicity, toxicity, and physicochemical properties. Pentapeptide WGPRL and tetrapeptide WLRL exhibited optimal ACE inhibition, deemed non-competitive. ADMET characteristics indicated good water solubility and human intestinal absorption, suggesting potential as peptide drugs or functional food ingredients. The impact of a novel antihypertensive peptide on Ang II-induced vascular smooth muscle cell dysfunction was explored. WGPRL and WLRL significantly inhibited Ang II-induced vascular smooth muscle cell over-proliferation and migration. These findings indicated that the potent antihypertensive SPH was enriched with a novel pentapeptide WGPRL and has potential as effective components of antihypertensive peptides functional food.