This report presents the instruction manual of the new water stress plugin developed by FAO in collaboration with the Stockholm Environment Institute's U.S. Center (SEI) for the calculation of the SDG indicator 6.4.2 “Level of water stress: freshwater withdrawal as a proportion of available freshwater resources” by river basin.Since the indicator was introduced in 2015, it has been used widely to estimate the level of water stress experienced at the country or regional level. With this new plugin, countries will be able to assess SDG 6.4.2 at the basin and sub-basin levels providing a different and more hydrologically sound view on the dynamics of water resources and their use. The plugin allows exploring the spatial and interannual trends of the level of water stress within a basin avoiding any multiple counting of its freshwater resources and taking into consideration the needs of water supply of the different sections of the basin. By supporting the improvement of water monitoring and management, this report contributes to the achievement of SDG 6.