Casein is a family of related phosphoproteins. Denaturation of casein was observed with the selected probiotic microorganisms, which can produce an enzyme dipeptidyl peptidase-4. Dipeptidyl peptidase-4 enzyme is the enzyme which helps in the denaturation of casein through probiotic microbes L. delbruekii sub sp bulgaricus and L. acidophilus. Probiotics' potential health and nutritional benefits have boosted the demand for functional probiotic foods. The efficacy of probiotics depends on providing a specific number of viable cells for their consumption. In the study enzyme secretion plays the main factor to show the viability of probiotic microorganisms to denaturate casein protein. HPLC-High-Performance Liquid Chromatography was conducted to observe the enzyme secretion. It was observed that the strain Lactobacillus delbruekii sub sp. bulgaricus had produced the dipeptidyl peptidase-4 enzyme at a higher amount of 174.841sum with a retention time of 14.27. The second probiotic strain Lactobacillus acidophilus resulted by producing enzyme Dipeptidyl peptidase-4 at 163.786 sums with a retention time of 13.209, The third strain and general probiotic strain Streptococcus thermophilus resulted dipeptidyl peptidase-4 enzyme production was not observed; hence denaturation of the milk protein was not resulted, due to the absence of the enzyme production.