The size, shape, and application techniques of gold leaf in fourteenth-and fifteenth-century gold ground panel paintings attributed to artists working in Florence, Siena, and Fabriano was directly visualized using in situ scanning macro X-ray fluorescence spectroscopy (MA-XRF). The resulting gold (Au) Lα (9.712 keV) element distribution maps are of sufficient spatial resolution to enable an accurate and reproducible measurement of the shape and size of individual gold leaves. The resulting leaf measurements are discussed in the context of historical guild regulations, in particular, the 1403 reforms to the statutes of the Florentine Guild of Doctors and Apothecaries (Arte dei medici e speziale), which standardized the dimensions of gold leaf produced in Florence. The dimensions of the gold leaf from Florentine paintings created before and after the 1403 reforms were compared to each other as well as to gold ground paintings created in Siena and Fabriano. The results revealed the gold leaf squares in fourteenth-century Florentine and Sienese panels had side lengths averaging about 8 cm. In contrast, the gold leaf squares used in the fifteenth-century Florentine and Sienese paintings were smaller, with side lengths measuring about 7 cm. In addition, the degree of overlap between adjacent gold leaves was also measured. The amount of overlap was found to vary between artists, and the degree of overlap was consistent within the oeuvre of a specific artist. Taken together, these results suggest that the dimensions of the gold leaves found in panel paintings relate to the place of production on the Italian peninsula and the period in which they were created, while the degree of overlap relates to the individual hand of a gilder or artist/ workshop.