The assessment of ecosystem services (ESs) plays a vital role in spatial planning and land-use decision-making. While ecosystems provide both tangible and intangible benefits essential for sustaining life, land-use changes driven by spatial planning influence the distribution and health of ecosystems, thereby affecting ES provision. To effectively identify priority areas for ES supply, it is essential to analyze ES hotspots, i.e., clusters of areas where individual ESs or bundles of ESs are delivered at high levels. Within this context, this study introduces a methodological approach for delineating ES hotspots. The first step involves a biophysical assessment of ESs, which here focuses on three regulating ESs: habitat quality, reflecting ecosystems’ capacity to provide niches and nurseries for wildlife; carbon storage and sequestration, indicating their role in climate mitigation; and land surface temperature, serving as a measure of local temperature regulation by vegetation and unsealed soils. In the second step, various spatial statistical techniques for hotspots identification are integrated to reduce the sensitivity associated with relying on a single method. The proposed approach is applied in the Italian regions of Campania and Sardinia, revealing significant differences in the extent and the spatial distribution of areas that serve as hotspots for all three ecosystem services. The proposed approach to ES delineation can be readily applied in any context where ES biophysical assessments are available. Besides aiding the identification of key areas for ES supply, it provides planners and decision-makers with robust, easily communicable information that can be effectively understood by a broader audience.