A Japanese painting on paper, in the form of a horizontal scroll from the Stibbert Museum in Florence, is being treated at the ISCR Paper Conservation Laboratory. The scroll, entitled Bamo Dōi-zu represents 33 different types of horses. Although there is no author's seal, it has been attributed to the famous Japanese artist, Kanō Sansetsu (1589–1651). Before conservation treatment, the artefact underwent technical and scientific examination in order to identify its materials and determine its conservation condition. As sampling paint layers from paintings on paper is extremely invasive, non-destructive techniques such as X-ray fluorescence spectroscopy, Raman spectroscopy, Fourier transform infrared spectroscopy, and spectrophotometry were employed. Several points from the paper support were analysed: the metal-leaf decorations, the seals and calligraphy, as well as the painted horses. Results detected and revealed a very simple palette: cinnabar (or its synthetic equivalent vermillion), shell white, carbon black, and the organic yellow gamboge. Pure gold and silver leaves were used for decorating the frontispiece. Useful information was obtained from the paper support and on its conservation conditions. Micro-destructive analyses were also performed on a few paper fragments for microscopic and scanning electron microscope equipped with an X-ray probe analysis. Results showed that materials and techniques are consistent with the Kanō School period.