Two pairs of hand-painted Japanese folding screens were analyzed by Raman, energy dispersive X-ray fluorescence (EDXRF) and Fourier transform infrared (FTIR) spectroscopy, in order to characterize the materials used in their production. Japanese folding screens, called byobu, are one of the oldest and most highly refined forms of Japanese art, where paper and silk were used as materials for the artists to paint on. The two pairs of folding screens studied in this work also exhibit a golden background to create a luminous effect. These screens depict some of the Portuguese who arrived to Japan during the 16th century, initiating active commercial and cultural exchange between Japan and the West, called the Nanban Trade. Nowadays, only about 60 examples of this Namban genre remain, so the study of these two pairs is of utmost importance to the knowledge of this precious craft. The materials identified, such as gold, silver, malachite, azurite, vermillion, red lead, red madder, yellow ochre, white oyster shell, and carbon black, are part of the traditional Japanese palette.