This paper illustrates a study for the reuse of selected historic buildings in the Medieval City of Rhodes in Greece. This study contributes to the understanding and interpretation of the chronological sequence of major intervention programs for the preservation of the Medieval City. The main idea of the project is the spatial distribution of compatible and various uses to reveal the unique character of the Medieval City. Spatial planning is proposed within the preserved urban zones in order to optimize and harmonize the selected uses according to the needs of sustainable urban development. Criteria to assess the compatible uses that ensure the features and architectural characteristics of the preselected historic buildings, located within the city fabric, were established and validated. The developed methodology that is presented herein and is an indispensable part of a pilot project may be applicable to other cases concerning historic cities. Finally, this paper aims to present a pilot program which promotes the reuse of historic buildings as a part of an integrated preservation plan. Inter-disciplinarity has set the basis for effective policies to guide and control the proposed pilot program, with ultimate objective to ensure sustainable preservation of the Medieval City of Rhodes.