In this chapter, I review the major interests and the challenges faced in the economic valuation of ecosystem services (ES) loss induced by transport infrastructure projects. First, infrastructure impacts on ES may be characterized for each ES and ecosystem types, including the characterization of the spatial extent of impacts and the magnitude of the impact. Not considering these issues may result in the underestimation of the economic loss engendered. The temporal trend of the ES values may also be addressed. The integration of ES loss into the project trade-off tools finally brings about other issues. For example, the analysis should, as far as possible, be exhaustive and discriminating; however, the precision of spatial models for the assessment should be balanced with the possibility of reproducing the analysis for multiple transport infrastructure projects. I further provide an example of the consideration of multiple ES loss, expressed in monetary terms, in a real Environmental Impact Assessment (EIA) and cost–benefit analysis (CBA) to assess the additional information it may bring. The analysis is based on the examination of a contemporary infrastructure project in France. The advantages can be summarized in three main points. Specifically, the analysis can (1) enhance guidance for avoidance measures from preliminary environmental studies to the comparison of implementation options, (2) provide the possibility of testing the cost-effectiveness of some mitigation measures, and (3) give an order of magnitude of the social loss engendered by the final option chosen. This analysis may enable a more efficient control of the natural capital loss in transport infrastructure planning. Finally, a description of the remaining research needs is presented.