Prestress force dominates the carrying capacity of concrete girders, and is vital for bridge health monitoring. Many vibrational-based methods have been proposed to determine prestress using bridge responses induced by known excitations, which means that they can barely use the normal traffic of in-service bridges as excitation to achieve long-term monitoring. Moreover, most studies are based on beam theory, which may not be precise for plate-like bridges. Hence, this paper establishes a motion equation for a prestressed slab via Kirchhoff’s plate theory and proposes a two-step procedure to assess the prestress and general excitation simultaneously through only bridge responses. The excitation is determined in the first step via the Load Shape function method and used as input for the prestress identification via state-space formulation in the second step. A numerical study on a prestressed plate subjected to a moving load is conducted. Considering different levels of measurement noise and load speed, the proposed method can determine the prestress and moving load with 7.33% and 10.18% error, respectively. A laboratory test on a prestressed box girder subjected to a fixed cyclic load is performed, the prestress and cyclic load are both determined to have good stability, and the errors are under 11.32%.