Testing methods are required for estimating prestress losses in Prestressed Concrete (PC) girder-bridges. They mainly include destructive approaches which cause significant damages. Conversely, dynamic nondestructive methods are unsuitable. Given these findings, a novel method for identifying residual prestress force in simply supported PC girder-bridges was implemented. Following the vertical load application in a three-point bending, the method estimates the prestress force by measuring the vertical deflection at a quarter or, alternatively, at the midspan of the PC girder-bridge. The method also requires information regarding its flexural rigidity. Particularly, the initial tangent Young’s modulus must be evaluated by compression tests on cores drilled at its quarter and midspan cross-sections after three-point bending. In absence of the geometric and/or material properties, the flexural rigidity can be estimated according to free vibrations. Secondly, the method comprises a reference solution, or a finite element model of the PC girder-bridge, in which the prestress force is unknown. Thirdly, the measured deflection becomes a parameter of the prestress force identification process. Accurate identifications are obtained when the deflection, under a higher vertical load, was precisely measured and the flexural rigidity was determined using reference solution and initial tangent Young’s modulus. In this article, the novel method was simulated on a simply supported PC beam-bridge subjected to time-dependent prestress losses for ≈9.5 months in the laboratory.