In recent years, the need for changes in the current consumption and development patterns has become evident. To achieve this, it is necessary to innovate, create, and devise new methodologies and ways of thinking that allow for a reorientation towards economically prosperous, socially equitable, and environmentally reasonable practices. The purpose of this study is to identify sustainability variables within the framework of the innovation system concept, and to propose a methodology for diagnosing regions and identifying their gaps in a sustainability-oriented innovation system. The methodology was based on a literature review of different documents, where sustainability variables related to innovation were extracted from this search, consisting of the identification of concepts made by different authors regarding what should be considered an innovation system aligned with sustainable development. These concepts were identified as study variables and a questionnaire was formulated based on them, which was reviewed by experts to determine their relevance and congruence. After obtaining the final questionnaire, which was subsequently referenced as the diagnostic tool, it was applied in a Colombian study region consisting of the states of Quindío and Risaralda, with sustainability stakeholders. As a result, a great lack of knowledge on the subject on the part of the respondents, low accessibility to sources of information, and a high percentage of disarticulation between policies were found, which led to the conclusion that the evaluated region has a low understanding of sustainability. Based on the information discussed in this research, it can be concluded that there are currently no specific methodologies to measure the sustainability of a region or territory. Therefore, the tool is determined to be a guide for the measurement of sustainability in the context of innovation in any region. Finally, from the studies reviewed, the potential to include sustainability in the innovation systems of a region was detected, enabling economic development, the production of goods and services, and strengthening the socio-environmental considerations involved in the adequate use of natural resources and the increase in the quality of life.