An important way to reuse agroindustrial by-products and to produce added-value products consists of the production of protein hydrolysates. In the current study, we used Brewer's spent grain (BSG), mainly because of its availability and cost, as a substrate for the enzymatic hydrolysis by Bacillus cereus. First, the physicochemical and microbiological characterization of BSG batches from three varieties was carried out. Furthermore, the optimal fermentation upstream processes for enzymatic hydrolysis by B. cereus were defined. Finally, the ability of B. cereus to hydrolyze different fractions of BSG was analyzed and possible synergistic effects of this bacterium along with other proteolytic bacteria were also investigated. Results showed that the naturally associated microflora was predominantly thermophilic aerobic bacteria and the drying process was the better alternative for BSG preservation. Water, lipids, and ash content differed significantly among the three varieties; however, no statistically significant differences were found in the protein content among them. After BSG characterization studies, the following protocol was set to obtain the fermentation substrate (FS): drying at 60°C for 24-48 H; sieving, grinding, and polyphenol extraction with an alcohol-water solution; and finally autoclaving. A synergistic effect was observed when B. cereus was inoculated with Pseudomonas strains in FS.