Fatigue is a common failure mode of steel bridges induced by truck traffic. Despite the deterioration caused by environmental factors, the increasing truck traffic volume and weight pose a premier threat to steel highway bridges. Given the uncertainties of the complicated traffic loading and the complexity of the bridge structure, fatigue evaluation based on field measurements under actual traffic flow is recommended. As the quality and the quantity of the available long-term traffic monitoring data and information have been improved, methodologies have been developed to obtain more realistic vehicular live load traffic. A case study of a steel interstate highway bridge using microscopic traffic simulation is presented herein. The knowledge of actual traffic loading may reduce the uncertainty involved in the evaluation of the load-carrying capacity, estimation of the rate of deterioration, and prediction of remaining fatigue life. This chapter demonstrates a systematic approach using traffic simulation and bridge health monitoring-based fatigue assessment.