The purpose of the article is to present the author’s methodology for identifying and assessing potentials and barriers occurring in various locations related to cultural tourism. The research aimed to develop a methodology to identify barriers and potentials for tourism development in peripheral and less-known locations. The potentials and barriers identified in a given location are important determinants of the development of cultural tourism and therefore economic development in the mentioned area. The identification of development potentials and barriers refers to the existing features of a given area or place and factors that activate or limit the possibilities of its using cultural resources. The study was based on the analysis of eight case studies of destinations with interesting cultural values. The entire series of analyses addressed various aspects of these destinations. Areas of different sizes and scales were considered, including areas in transboundary regions, rural areas, historic city centres, and archaeological or industrial sites. The work presents the author’s methodology for integrating the various elements that define the features and factors of cultural tourism and establishes a framework for a strategy for its sustainable development. To achieve this, a tool in the form of a comprehensive matrix was developed to assess the situation of each pilot site. Based on this tool, the pilot sites identified key features or factors as a barrier or potential and furthermore defined their territorial scope and their impact on their chosen dominant types of tourism. Barriers and potentials were identified based on a set of 47 previously developed features and factors. During the research, 38 most important potentials and 15 barriers were identified. The tool presented in the paper is comprehensive and universal, so it can be applied to any destination.