The article focuses on the biotechnological valorisation of brewer's spent grain (BSG) for the production of high-value compounds, specifically hydrogen as a clean energy alternative. BSG, a byproduct of the beer industry rich in fiber and protein, presents an opportunity for sustainable waste management and energy generation. Through a thermal-hydrolysis process optimized using statistical methods, the authors aimed to extract reducing sugars and proteins from BSG for subsequent conversion into hydrogen. The study utilized Escherichia coli in fermentation processes to analyze the conditions leading to the highest hydrogen production, along with volatile fatty acids and ethanol. The research highlighted the significance of temperature and acid concentration in the hydrolysis of BSG, showcasing the potential to achieve high concentrations of reducing sugars and proteins under optimized conditions. By identifying the most effective fermentation conditions, such as 117°C, 20 minutes, and 0.1 M H2SO4, the study successfully generated 48 mmol/L of hydrogen, demonstrating the feasibility of utilizing BSG for sustainable energy production. Overall, the findings emphasize the value of biotechnological approaches in converting agro-industrial waste like BSG into valuable products, thereby addressing environmental concerns and promoting the shift towards cleaner energy sources. The study contributes to the ongoing efforts in developing sustainable practices in the production of biofuels and highlights the potential of BSG as a renewable resource in the transition towards a more environmentally friendly energy landscape.