Bioactive peptides can be produced from milk proteins in fermented products by proteases of lactic acid bacteria. The cell envelope protease (PrtS) of Streptococcus thermophilus is anchored at the cell wall, but we recently discovered that the 4F44 strain produces a soluble form that can be recovered in medium supernatant. This work was aimed at optimising the production of bioactive peptides from bovine caseins. By growing S. thermophilus 4F44 in the newly designed YLUNi medium, a high quantity of the soluble form of PrtS could be produced that could be directly used as the proteolytic agent on sodium caseinate. Peptide production was monitored by reverse phase-high performance liquid chromatography and tandem mass spectrometry; of 247 peptides identified, 143 were derived from β-casein. Twenty-two peptides, already reported in the literature as bioactive, include ACE-inhibitory, antioxidant, immunomodulating, or antibacterial peptides; addition of such peptides could improve the health benefits of dairy products.