In the Lower Mekong River Basin (LMB), deforestation rates are some of the highest in the world as land is converted primarily into intensive agriculture and plantations. While this has been a key for the regions economic development, rural populations dependent on the freshwater water resources that support their fishing and agriculture industries are increasingly vulnerable to the impacts of flood, drought and non-point source pollution. Impacts of deforestation on ecosystem services (ES) including hydrological ES that control the availability and quality of fresh water across the landscape, regulating floods and droughts, soil erosion and non-point source pollution are known. Despite this understanding at the hillslope level, few studies have been able to quantify the impact of wide-scale deforestation on larger tropical watersheds. This study introduces a new methodology to quantify the impact of deforestation on water-based ES in the LMB with a focus on Cambodia by combining spatial datasets on forest loss from remote sensing and spatially-explicit hydrological modeling. Numerous global and regional remote sensing products are synthesized to develop detailed land use change maps for 2001 to 2013 for the LMB, which are then used as inputs into a hydrological model to develop unique spatial datasets that map ES changes due to deforestation across the LMB. The results point to a clear correlation between forest loss and surface runoff, with a weaker but upward trending relationship between forest loss and sediment yield. This resulted in increased river discharge for 17 of the 22 watersheds, and increased sediment for all 22 watersheds. While there is considerable variability between watersheds, these results could be helpful for prioritizing interventions to decrease deforestation by highlighting which areas have experienced the greatest change in water-based ES provision. These results are also presented in a web-based platform called the Watershed Ecosystem Service Tool.