The Mississippi Delta, a portion of the Mississippi Alluvial Plain (MAP) located in northwest Mississippi (USA), is an area dense with industrial-level agriculture sustained by groundwater-dependent irrigation supplied by the Mississippi River Valley alluvial aquifer. Observed declines in groundwater-level elevations and streamflow, contemporaneous with increases in irrigation, have raised concerns about future groundwater availability and the effects of groundwater withdrawals on streamflow. To quantify the impacts of groundwater withdrawals on streamflow and increase understanding of groundwater and surface-water interaction in the MAP, hydrograph-separation techniques were used to estimate baseflow and identify statistical streamflow trends. The analysis was conducted using the US Geological Survey Groundwater Toolbox open-source software and daily hydrologic data provided by a spatially distributed network of paired groundwater wells and streamgage sites. This study found that statistically significant reductions in stream baseflow occurred in areas with substantial groundwater-level declines. The use of hydrograph-separation and trend analyses to quantify the impacts of groundwater withdrawals and the use of streamflow as a proxy for changes in groundwater availability may be applicable in other altered environments. Characterizing and defining hydrologic relations between groundwater and surface water will help scientists and water-resource managers refine a regional groundwater-flow model that includes the Mississippi Delta, which will be used to aid water-resource managers in future decisions concerning the alluvial aquifer.