In recent decades, many works have been devoted to the study of materials and painting techniques used to produce illuminated manuscripts. If the analyses were once largely invasive, the approach has become increasingly more in situ and non-invasive over the years. This work presents the results of the analysis of the Portuguese Charter of Évora, an illuminated manuscript that dates back to 1501, combining an elemental mapping technique (MA-XRF) with the non-invasive imaging techniques of infrared reflectography and digital microscopy. Remarkably, this approach allowed us to obtain unexpected results regarding the chronology of production of the illumination of the view of the City of Évora and of the Charter of Évora itself, posing new questions for art history on the political, social and artistic context of the early 16th century City of Évora.