One of the crucial steps for a successful integration of electric bus fleets into the existing electric power systems is the active and intelligent usage of their flexibility. This is important not only for reducing the eventual negative effects on the power grid but also for reducing energy and infrastructure costs. The first step in the optimal usage of flexibility is its quantification, which al-lows the maximum provision of flexibility without any negative effects for the fleet operation. This paper explores the available flexibility of large‐scale electric bus fleets with a concept of centralized and unidirectional depot charging. An assessment of available positive and negative flexibility was conducted based on the data from two real bus depots in the city of Hamburg, Germany. The analysis shows the biggest flexibility potential was in the period from 16:00 h to 24:00 h, and the smallest one was in the periods from 08:00 h to 16:00 h, as well as from 02:00 h to 08:00 h. The paper also gives an overview of the possible markets for flexibility commercialization in Germany, which can provide an additional economic benefit for the fleet operators. A further analysis of the impact of parameters such as the timeline (working day or weekend), charging concept, ambient temperature, and electrical preconditioning provides an additional understanding of available flexibility.