This paper describes a methodology and scenario-based software toolbox for the analysis, design and optimization of electric bus operations. The software toolbox contains all relevant alternative propulsion concepts and enables the identification of a cost-optimal solution for planning the necessary charging and refueling infrastructure. The toolbox has a modular structure and enables an investigation adapted to the boundary conditions of the bus network. The current procedure for identifying new locations for charging infrastructure is very manual. In a complex system, manual analyses hardly lead to a cost-optimized solution. During the optimization with the developed toolbox, a heuristic and rule-based greedy optimization algorithm is used to determine f.e. the minimum number of electrified stations and the number of required charging points. Furthermore, the infrastructure planning includes the analysis of the required grid connection to supply the infrastructure with energy. Two case studies present selected results from the application of the software toolbox. For a rural bus operation in Germany, the operation of electric buses was investigated and compared. Depending on the ambient temperature (−20°C to +40°C), different energy consumptions in bus operation could be determined. In this study, the energy consumption of a 12m solo battery bus is between approx. 1.4 kWh/100km and 4.3 kWh/100km. The optimization of electrified stations is presented in the second case study. In a system with hundreds of rotations and almost 100 end stations, in the base setup 77 stations were identified that enable electrified operation. Through the optimization based on the software toolbox, the number of required stations could be reduced by approx. 35% to 50 stations compared to the manual variant.