The centroid of stiffness is a universal parameter and its position in a cross-section can be evaluated for any load-carrying beam structure as the position of the neutral axis under conveniently chosen loads. Thus, a change in the position of the neutral axis within a cross-section can indicate change in the position of the centroid of stiffness, i.e., unusual structural behaviors. This study presents a probabilistic damage detection method based on changes in the position of the neutral axis. Therefore, the method presented in this paper is potentially applicable to a large variety of beam-like structures. Dynamic strain data from the US202/NJ23 highway overpass in Wayne, NJ, is used to assess and validate its performance. The results show that the neutral axis location is varying even when damage is not present. Reasons to this variation are proposed and the accuracy in the evaluation assessed. The conclusion of this study is that the position of the neutral axis can be evaluated with sufficient accuracy using dynamic strain measurements and presents its potential to be used as a damage sensitive feature.