Soybeans are the most popular source of vegetable protein for the people of Indonesia, having protein content ranging from 35%-38%. In the protein analysis of 100 mesh size soybean powder using the Kjeldahl method with the Kjeltec 2100 Distilling Unit, the protein content is 35.95. The high protein content of soybeans can be used for the manufacture of Peptone by hydrolysis process using the papain enzyme. This study used a ratio of 1:2 substrate concentration, 0.3% enzyme concentration, pH 5-7, and a temperature of 40 °C-60 °C. The results of this study obtained the highest total dissolved nitrogen value of 14.7264%, at a temperature of 50 °C, pH of solution 5 and hydrolysis time of 7 h. The total dissolved nitrogen value was compared with the total nitrogen value of the material as a value indicating the result of enzymatic hydrolysis. Peptone-hydrolyzed soybeans are used as an alternative medium for the growth of Escherichia coli ATCC 25922 and Candida albicans ATCC 1023.