Prestress losses have a direct impact on concrete stress development and deflection behavior of highway bridge members. A poor estimate of pre-stress losses can result in a structure in which allowable stresses are exceeded or camber and deflection behavior is poorly predicted, such that the serviceability of a structure may be adversely affected. This paper reports the prestress losses observed throughout fabrication, shipment, erection, and the first 2 years of service for the first high-performance superstructure concrete bridge in Missouri. The prestress losses investigated included prerelease losses, elastic shortening losses, relaxation losses, creep losses, and shrinkage losses. Results from the study were compared with eight commonly used loss estimate models for total prestress losses, including AASHTO and Prestressed Concrete Institute methods. Recommendations were proposed by the authors for the most appropriate methodology to use to predict prestress losses in high-strength concrete girders accurately.