The objective of the research study was to determine the amino- and dipeptidase activity of selected lactic acid bacteria cultures (Lactobacillus casei, Lactobacillus acidophilus) subjected to heat- treatment at temperatures ranging between 50 and 75 °C for 1, 15, and 25 minutes. The analyzed Lactobacillus cultures synthesized peptidases that had a similar substrate specificity but different activities. They showed a higher activity of aminopeptidases compared to the activity of dipeptidases. The mean dipeptidase activity of Lb. acidophilus was 45% higher than that of Lb. casei. The Lb. casei strain was characterized by a 25% higher activity of aminopeptidases. This strain exhibited the highest specificity to the AlaLeu, Ala-Ala, and Gly-Leu substrates, whereas the Lb. acidophilus strain – to the Ala-Ala and Ala-pNA substrates. The highest activity of the amino- and dipeptidases of Lb. casei and of the dipeptidases of Lb. acidophilus was reported after the bacteria were heat-treated for 15 min. As for the amino- and dipeptidases of Lb. casei, their activity was, respectively: 5,10, and 0.83 U/min/mg, and as for the dipeptidases of Lb. acidophilus, it was 1.66 U/min/mg. The mean aminopeptidase activity of Lb. acidophilus increased along with the increasing heat-treatment time; its highest values were reached after 25 min (3.89 U/min/mg). It was proved that the increasing of the heat-treatment time significantly impacted the growth of aminopeptidase activity of lactic acid bacteria; this fact confirms their high temperature resistance.