Fatigue is a common failure mode for steel bridges. About 80-90% of failures in steel structures are related to fatigue and fracture. Despite the deterioration caused by environmental factors, the increasing traffic volume and weight pose a premier threat of steel highway bridges. The total number of truck passages in the 75-year life of a highway bridge could exceed 100 million. With the aging of existing steel highway bridges and the accumulated damage under truck loading, the fatigue assessment for continuing service has become important for decisions making on the structure maintenance, component replacement, and other major retrofits. This research seeks to develop a framework for the fatigue assessment of steel highway bridges based on simulated truck loading. The I-270 Bridge over Middlebrook Road was numerically studied with the proposed methodology. With the help of the available long-term monitoring traffic data and information, truck loading was obtained through the probability-based full velocity difference model. Then, the three-dimensional finite element (FE) global and local bridge models were studied subjected to the simulated truck loading. Meanwhile, the preliminary field test and the long-term monitoring test were also been conducted. The FE models were calibrated by the collected field measurements through monitoring systems, and the simulated numerical structural responses were validated. Lastly, Miner's rule and the rainflow counting algorithm were used in the analysis of simulated numerical structural responses to estimating the fatigue life. Thus, the proposed methodology could be used to realistically simulate the fatigue behavior of steel highway bridges under current or future truck loading, to direct the experimental designs and instrumentation plans before performing experiments on laboratory or on site, and to better understand the fatigue mechanism and prevent the fatigue damage of steel highway bridges.