Abstract A recent restoration of the mosaics of the Baptistery in Florence enabled the collection and analysis of a group of glass tesserae. Twenty-three tesserae, dated between the end of the 13th century and the first half of the 14th century, showing different colors were sampled and analyzed to identify the raw materials, colorants and opacifiers employed in their production. Chemical analyses of major and minor elements, performed by WDS-EMPA, showed that the sample set can be subdivided into two groups on the basis of the flux employed during production: i) a group of seven samples with high levels of K 2O, and ii) a group of sixteen Na-rich samples. Analyses of the main components of the glass, yielded by the vitrifying portion, suggest that two different typologies of raw materials – with different proportions of carbonatic and feldspatic components – were employed for the production of these tesserae. Scanning electron microscopy observations, coupled with EDS qualitative analyses and X-ray powder diffraction experiments, revealed the presence of SnO 2 crystals dispersed in a high lead matrix in all the opaque samples, with higher concentrations of particles in the white samples. Spectroscopic analyses using Fibre Optic Reflectance Spectroscopy (FORS) for the identification of chromophores were also conducted on the mosaic tesserae with the aim of acquiring data for integration with information collected in other analyses. This technique was applied in situ, being non-invasive, simple and inexpensive. The data here obtained are compared with the historical sources available (glass recipe books) to identify the raw materials and the glassmaking techniques employed in view to establish relationships between the existing local production and the analyzed mosaic glass.