The hydrolysis of whey protein isolate with low purity enzymes was studied with application of ultrasound pretreatment. The aim was to increase the degree of hydrolysis and produce new biologically active peptides. The experiments were carried out with hydrolysis times up to 24 h. The influence of temperature, pH, enzymes (pepsin and papain), substrate concentration, and use or not of the ultrasonic probe was investigated. The peptide’s structure and profile were examined after exposure to ultrasound. The highest degree of hydrolysis occurred with the use of pepsin for 10 h, 37 degrees C, and pH 2.5. The ultrasound pre-treatment allowed a reduction of 6 h in the process. After partial enzymatic hydrolysis and ultrasound pretreatment a higher proportion of low molar mass peptides were observed at 1000-2000 g.mol(-1). In vitro simulated digestion showed slight differences in peptide profile indicating that active compounds were preserved intact with a slight impact on bioactivity.