Tenderness of meat is one of the most important features of culinary beef. It is primarily created during the aging of meat. This long-term process with endogenic enzymes participating in it may be accelerated using egzogenic proteolytic enzymes. The objective of the present study was to assess the usefulness of two enzymatic preparations (YG and YO), obtained from a culture of Yarrowia lipolytica yeasts (strain PII6a) and applied to tenderize beef, as well as to compare their effectiveness with the effectiveness of pepsin. The experimental material was enzymatically hydrolyzed using some selected preparations at two temperatures: 4°C and 18°C, for 6 and 24 hours, and at two pH values of the environment (3.5 and 6.0). The effect of the experimental enzymes was assessed on the basis of the degree of meat protein degradation. The latter was determined by the increase in the content of free amine groups, hydroxyproline, and nitrogen contained in post-hydrolysis solutions, as well as on the basis of measured levels of meat tenderness using special gauges. It was stated that the enzymatic preparation ‘YO’ showed the highest proteolytic activity if compared with the proteins of beef. The pH acidity of the environment equalling 3.5 was optimal for the activity of the assessed enzymatic preparations; the temperature did not have any relevant impact on the protein degradation process. It was proved that a prolonged hydrolysing process, from 6 to 24 hours, had a significant effect on the improvement of meat tenderness although the degradation was the most intensive during the first 6 hours.