Recently, Structural health monitoring (SHM) algorithms based on the modal flexibility matrix has drawn significant attention. These algorithms have been shown to be more effective in damage localization than other methods such as using natural frequencies or mode shapes alone. However, not many of these algorithms are directly applicable for inservice highway bridges. Some studies that are done on highway bridges use SHM algorithms that require known excitations. Other studies require the use of a finite element model to implement the flexibility-based approach. In this paper, a model-free modal flexibility based SHM algorithm is used to identify structural damage while compensating for variations in modal properties due to temperature fluctuations. The algorithm is first validated using a laboratory scale girder bridge under constant temperature. The modal flexibility of different damage scenarios is compared to a baseline undamaged state. Finally, the efficacy of the SHM algorithm is verified using data collected under ambient loading conditions on an in-service highway bridge.© (2011) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.