According to the Water Framework Directive, the Ecological Flow (Eflow) is assumed to be the minimum water discharge required to achieve and maintain the environmental objectives of “good quality status” in a natural water body. It is highly recognized that, the hydrological regime of natural flow plays a primary and crucial role influencing the physical conditions of habitats, which in turn determines the biotic composition and sustainability of aquatic ecosystems. Furthermore, the simple assumption to supply a minimum instream during dry periods is not enough any longer in order to protect the river environment. The recent hydro-ecological understanding states that all flow components might be considered as operational targets for water management, starting from base flows (including low flows) to high and flood regimes in terms of magnitude, frequency, duration, timing and rate of change. Several codes have been developed and applied on different case studies in order to define common tools to be implemented for the Eflow assessment. The study proposes the application of the Indicators of Hydrologic Alteration methodology (IHA by TNC) coupled to the valuation of the Index of Hydrological Regime Alteration (IARI by ISPRA) as an operative tool to define the ecological flow in each monitoring cross section to support the sustainable water resources management and planning. The case study of Agri River, in Basilicata (Southern Italy) is presented. The analyses have been carried out on monthly discharge data derived applying the HEC-Hydrological Modelling System at the basin scale using the daily rain data measurements obtained by the regional rainfall gauge stations and calibrated through the observed inlet water discharge registered at the Lago del Pertusillo reservoir station.