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The paper investigates the occurrence of relaxation processes in enzymatic hydrolysis of whey proteins in phosphate buffer using high-resolution ultrasonic spectroscopy. The hydrolysis produces a frequency-dependent evolution of ultrasonic velocity and attenuation originated by the proton transfer between the phosphate ions and the terminal − N H 2 group of protein hydrolysates. Such frequency dependence is absent in other systems, e.g., Tris buffer. Real-time profiles of ultrasonic velocity and attenuation measured during the hydrolysis of β–lactoglobulin, α–lactalbumin and bovine serum albumin by α–chymotrypsin were employed in quantitative analysis of the relaxation mechanisms, relaxation times, determination of the proton transfer rate constants and the reaction volume. The proton transfer is characterised by the rate constant 9.4 × 107 L mol−1 s−1 and the adiabatic reaction volume 24 × 10−6 m3 mol−1. The obtained reaction volume agrees well with thermodynamic data for ionisation volumes and enthalpies of the terminal amino group and phosphate. The described methodology for estimating the relaxation contribution to ultrasonic characteristics allows for precision real-time ultrasonic measurements of the concentration of peptide bonds cleaved during protein hydrolysis in a broad range of environmental conditions. [Display omitted] • Precision real-time monitoring of protein hydrolysis with HR-US. • Evolution of ultrasonic characteristics during hydrolysis is frequency dependent. • Relaxation processes accompanying protein hydrolysis were analysed. • Kinetics and thermodynamics of peptide-phosphate proton transfer was studied. • Ionic effects on kinetics and thermodynamics of the proton transfer were described. [ABSTRACT FROM AUTHOR]