Type 2 diabetes is considered a severe problem for the public sector around the world. DPP-IV inhibitors have shown a favorable therapeutic profile for glycemic control. This work aimed to evaluate the growth of two probiotics on a fermentation medium enriched with rye protein and determine the antidiabetic capacity from protein hydrolysis by these bacteria. Two strains were activated and adapted by cultivations in MRS broth and rye protein medium (7.5% rye isolate protein, 1% glucose, and solved in 0.1 M, pH=6.8 phosphate buffer). The last medium was used as a starter culture for fermentation systems, inoculating at 1% to the novel rye protein medium. Fermentation was performed at 37°C for 24 h, and the viable count was made afterward. Also, samples were centrifugated at 10,000 rpm at 4°C for 10 min to separate the supernatant, which was used to measure the hydrolysis degree and DPP-IV inhibition using spectrophotometric methods. The viable count in fermentation systems was 7.58±0.02 and 8.47±0.07 log CFU/mL for L. rhamnosus GG (LR) and L. casei Shirota (LCS) at 0 h. After 24 h fermentation, both strains exhibited a 2 logarithmic cycle growth, reaching 9.72±0.10 and 10.52±0.07 log CFU/mL. The LCS strain presented the highest nitrogen requirements because the free amino groups' concentration increased from 11.80±0.00 to 891.78±48.92 mg/L. At the same time, it was practically constant for LR, showing 163.33±6.97 and 167.50±1.54 mg/L at 0 and 24 h, respectively. In the case of DPP-IV inhibition, both strains displayed similar bioactivity, with LR increasing from 5.72±0.14 to 20.32±0.95%, and LCS from 10.37±1.04 to 27.04±1.57%. In conclusion, the rye medium allowed the growth of both strains to reach adequate levels for probiotics, and similar bioactivity was found in the fermentation systems tested, representing an excellent opportunity for developing fermented products from vegetable sources.