Sonoporation stimulates cell growth as it improves the permeability of the membrane and increases the uptake of impermeable molecules in the extracellular matrix. We evaluated the effects of substrates (whey, whole, and skim milk) and ultrasonic treatments (ultrasonication and thermosonication) on the antioxidant activity (AA) of water-soluble kefir postbiotics (WSKPs). The samples were evaluated in terms of antioxidant activity (ABTS, DPPH, FRAP, and ORAC), water-soluble protein content, proteolysis (SDS-PAGE profiles), and cell membrane permeability. The levels of AA in all WSKPs depended on the substrate and method of obtaining them. However, the WSKPs from whey had higher antioxidant activity with DPPH (11.11 mg TE/100 mL), ABTS (12.77 mg TE/100 mL), and FRAP (5.18 mg TE/100 mL). Also, the WSKPs from whey had the highest values for water-soluble protein (1.45–1.32 mg/mL) and proteolysis degree and the lowest percentage of dead cells (11.4–28%). These results suggest that the production of WSKPs from whey might add value to whey production. Furthermore, WSKPs have potential as a functional ingredient in the production of beverages or foods with antioxidant activity.