Not many manuscript maps have been the object of material analysis so far. A portolan chart, signed and dated by Juan Vespucci in 1520, was studied in this research, conserved at the Archivo General de Indias in Seville (Spain). It is made on parchment and depicts the coasts and islands of Europe and Africa. It is the oldest portolan chart made in Seville, being unusual in applying hand stamp for decorative figures. The map was analysed by different non-invasive techniques: infra-red and ultraviolet light, digital microscope and X-ray fluorescence (XRF). The main goals of this study were to identify the materials used, as well as to detect retouching or restoration work. Results showed that the entire parchment was first covered with a white layer made of lead white (Pb), calcite or gypsum (Ca). The principal pigments used were vermilion (Hg), yellow ochre (Fe), azurite (Cu) and a copper-based green pigment (Cu) and carbon black. The letters were probably written with an iron-gall ink (Fe, Cu). Very thin golden leaves were applied on a mixtion glue for gilded wind roses. Several retouches from the 19th/20th centuries were found using zinc and titanium whites and probably cobalt blue.