This study aimed to define the optimal composition of three heterogeneous substrates of the anaerobic digestion process to maximize methane production. The investigated substrates were sewage sludge (SS), the organic fraction of municipal solid waste (OFMSW), and horse waste (HW). The optimal composition of these substrates was defined using the mixture design and, more specifically, the simplex–centroid mixture design. Customized methods and materials were employed to study the complex mixture design of these substrates. The findings revealed that the optimal mixture involved all three substrates with the composition 0.17 HW, 0.66 SS, and 0.17 OFMSW, which demonstrated the highest methane yield at 269 NmL·gVS−1. In addition, a mathematical model was developed to predict methane production based on a specific composition of co-substrates. The results were validated at the small pilot scale.