Mega-urbanization is a major driving force of environmental changes and sustainability because of its speed, scale and worldwide connectivity. Megacities, due to their spatial extent, multicultural demographic structure and multifunctional land-uses, demand new sophisticated scientific approaches to meet the challenges posed by these socio-ecological complexities. Scientists studying megacities are always confronted with the challenge of stratifying their sampling sites for in-depth field investigations particularly in developing countries where the urban landscapes do not expand in line with any predetermined plans – nevertheless with paradigmatic phenomena. This challenge becomes more complicated when considering socio-ecological studies because of the complexity of coupled human–environmental systems where multifunctional composites of land-uses and respective land cover interplay. This paper presents a conceptual framework for developing an urban gradient model, adapted from a set of scientific postulates to systematically hypothesize the selection of research/sampling sites in the field in large urban agglomerations. This framework, due to its scientific legitimacy, is named the Theorized Urban Gradient (TUG). It is based on the assumption that it would allow examination of field/data samples from a variety of urban structures incorporating different functional characteristics. An application of the framework is presented for the megacity of Karachi, Pakistan, comparing the TUG with other standard sampling methods to test and to show the value and the advantages of it. A systematic selection of samples in a compact and informally growing urban landscape is justified. Results corroborate urban gradient, being supported by deductive scientific postulates, as an important research method rather than merely a modelling approach.