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Highlights • Multidimensional assessment of ES delivery integrating marine domains information. • Method to operationalize ES incorporation in ecosystem-based MSP of the Adriatic Sea. • Measurable ecological functions as indicators for supporting ES. • Hotspot-Richness analyses overlap indicates areas of high provisioning levels of ES. • Method for optimal zoning plans for sustainability and marine conservation actions. Abstract The assessment and mapping of ecosystem services (ES) has become an increasingly important instrument for environmental management and conservation priority-setting. As such, this practice can be used in ecosystem-based Marine Spatial Planning (MSP). MSP is recognized as an opportunity to achieve socio-economic and ecological goals simultaneously, to suggest solutions for sustainable management of marine environment and its resources. In this study, we propose an operational approach that includes novel spatial analysis in the marine field to quantify and map supporting ecosystem services. Such approach spans the 3D-dimension of the marine environment, considering all marine domains (sea surface, water column, seabed) separately. Our approach is focused on mapping supporting ES of the Adriatic Sea, to grant their preservation in order to guarantee the delivery of all other ES. Supporting ES provision in the Adriatic was quantified through the use of indicators that denote ES delivery and that are specifically related to the three marine domains. We identified areas of elevated provisioning levels of multiple supporting ES in the Adriatic, which is hypothesized to be priority areas of conservation. Our results confirm the importance of explicitly including the pelagic domain in planning and conservation processes. Areas that provide the lowest levels of supporting ES delivery were also mapped, to indicate possible 'sacrificial areas' for industrial or intensive use. The spatial coincidence of the determined hotspots areas of ES delivery associated with particular marine areas that are and are not under conservation regimes was analysed. This approach led us to test the applicability of the method for identifying marine areas for conservation purposes. Our methodological approach aims at producing relevant scientific knowledge for prioritizing marine conservation and sustainable management actions, to be used in MSP and marine management. [ABSTRACT FROM AUTHOR]