An increasing number of studies demonstrate the need of applying a social-ecological system approach for landscape planning. However, there is a lack of empirical research that operationalizes the concept of social-ecological system for landscape planning through the characterization of social-ecological interactions. In this study, we develop a methodological framework to delineate the boundaries of social-ecological systems and to characterize their main social-ecological units in a spatially explicit way. Social-ecological units represent the interactions between the biophysical and socio-economic sub-systems at local scale. The methodology is structured in four phases: (1) ecological regionalization, i.e. identification and mapping of consistent ecological units based on biophysical variables; (2) socio-economic regionalization, i.e. identification and mapping of homogeneous groups of municipalities based on socio-economic variables; (3) identification of social-ecological systems boundaries and characterization of social-ecological units; and (4) validation of the social-ecological systems boundaries with key informants through participatory mapping. By applying the proposed methodological framework to three different Mediterranean cultural landscapes, we define the boundaries of social-ecological systems and illustrate how social and ecological sub-systems interact at local scale. We conclude that the proposed methodological framework is useful to operationalize the concept of social-ecological systems in landscape planning.