Automated guided vehicle (AGV) transportation systems are an important component of flexible warehouse systems, and research on the performance evaluation of AGV transportation systems contributes to the optimization and decision-making of flexible warehouse systems. Compared to simulation and experimental methods, analytic methods require less computational time, which is more applicable when integrated with optimization and decision-making methods. However, the previous analytical methods for performance evaluation neglected the velocity change and collision avoidance of AGVs, which decreased the evaluation accuracy. Therefore, herein, a throughput evaluation methodology based on AGV route decomposition is proposed for AGV transportation systems. In the proposed methodology, an AGV transportation system is modeled as an open queueing network model considering the velocity change of AGVs. The open queueing network model is decomposed into sub-models based on preplanned AGV routes, considering the collision avoidance among AGVs, to decrease the analysis complexity and computational time. Finally, the throughput of the AGV transportation system is obtained by the throughput sum of the sub-models corresponding to the unload stations. The numerical results indicate that the proposed methodology evaluates the throughput of an AGV transportation system with an accuracy of ≥94%. This study will facilitate the efficient throughput and decision-making of AGV transportation systems.