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Using agricultural waste biomass and by-products (alternative substrates) for biogas production is a promising strategy to improve energy self-sufficiency in the European Union, and the sustainability of biogas production. A comprehensive study with three different types of alternative substrates was conducted on lab-scale. The methane potential of the untreated alternative substrates was 44.1–66.5% of maize silage. Promising substrate pre-treatment methods were identified, enhancing the methane potential by up to 46% and the degradation kinetics by up to 106%, respectively. Simulating the methane production in continuously operated digesters by parameters obtained in the batch tests was possible. High accuracy of the parameters was essential to obtain reliable simulation results. Applying the simulation approach to a case study revealed that the methane production is reduced by 36%–73% if the alternative substrates substitute maize silage. To analyze the economic feasibility, this was contrasted with prices for maize silage and electricity in Germany in 2022. Only maize straw showed the potential to replace maize silage during those hours that featured the 25% lowest electricity prices. [ABSTRACT FROM AUTHOR]