Although the benefits of the Lean Six Sigma (LSS) methodology have been proven for more than 20 years, it is still underutilized in environmental science, (e.g., in anaerobic digestion and biogas production). In order to obtain a structural and data-oriented perspective for process optimization in the renewable energy sector, LSS application must be considered one of the most valuable tools. To inform this paper, the LSS analysis phase was conducted in a scaled-down environment through detailed laboratory experiments. Results showed not only the feasibility of LSS application in biogas technology, but also some useful findings such as possible root causes for low production, such as impurities, waiting time, and existing pre-treatment methods for the defined problem. The results of the experiments show that the use of old substrates can reduce the biogas production up to half of the production with fresh substrates, and that even a 10% sand content can reduce the production up to 14.2%, which shows the need for a solution to these two issues.