Although the impact of hydrology on the ecological status of surface water bodies has been highly recognised, the hydrological regime alteration assessment has proven to be a challenging task. In this context, an extensive structured review analysis was used as a research method to investigate the strength and limitations of the hydrological regime alteration assessment methods as adopted by each member of the European Environment Agency and the cooperating countries, according to the Water Framework Directive 2000/60, as well as to propose future directions. The review was also widened to include the methods currently used worldwide in the hydrological alteration studies and the supporting software tools developed. The implementation of a common methodology on a European scale is not applicable, since a single approach would not be able to cope with the regional needs and conditions. The main limitation in almost all the methods developed by European countries and worldwide is the need for a flow time series of high temporal resolution, so as to also capture the systems’ extreme high and low flows. Automatic monitoring systems for rivers can provide a solution. Additionally, hydrological modelling may provide the necessary data for the definition of the reference conditions. Nevertheless, the main limitations of the methodologies reviewed and the challenge for future development are the incorporation of the groundwater contribution to the hydrological regime and the development of quantitative relationships between flow alteration and ecological response.