The need to further abate agricultural nitrate (N)-loadings to coastal waters in Denmark represents the main driver for development of a new spatially targeted regulation that focus on locating N-mitigation measures in agricultural areas with high N-load. This targeting makes use of the spatial variation across the landscape in natural N-reduction (denitrification) of leached nitrate in the groundwater and surface water systems. A critical basis for including spatial targeting in regulation of N-load in Denmark is the uncertainty associated with the effect of spatially targeting measures, since the effect will be critically affected by uncertainty in the quantification of the spatial variation in N-reduction. In this study, we used 30 equally plausible N-reduction maps, at 100 m grid and sub-catchment resolutions, for the 85-km2 groundwater dominated Norsminde catchment in Denmark, applying set-aside as the measure on high N-load areas to reach a N-load reduction target of 20%. The uncertainty on these N-reduction maps resulted in uncertainty on the estimated N-load and on the required set-aside area. We tested several methods for spatially targeting set-aside that took into account the uncertainty on set-aside area and developed methods to reduce uncertainty on the estimated N-load reductions. These methods includes application of set-aside based on each individual N-reduction map compared to a mean N-reduction map, using spatial frequency of high N-load and using spatial frequency of low N-reduction. The results revealed that increasing the ensemble size for averaging the N-reduction maps would decrease the uncertainty on the estimated set-aside area with a stable effect when using an ensemble of 15 or more maps. The spatial resolution of the groundwater N-reduction map is essential for the effectiveness of set-aside, but uncertainty of the finer spatial resolution of N-reduction is greater compared to sub-catchment scale, and application of a spatially targeted strategy with uncertain N-reduction maps will result in incorrect set-aside area and uncertain estimations of N-load reductions. To reduce the uncertainty on estimated N-load reductions, this study finds the method of set-aside application based on spatial frequency of high N-load to be more effective than other methods tested.