Three Japanese woodblock prints from the Edo period (1603-1868) underwent a scientific investigation with the aim of understanding the changes in the colorants used in Japanese printing techniques. A multi-analytical approach was adopted, combining non-invasive techniques, such as fiber optic reflectance spectroscopy (FORS), Raman spectroscopy, multispectral imaging (MSI), and macro X-ray fluorescence (MA-XRF) with minimally invasive surface-enhanced Raman spectroscopy (SERS). The results enabled many of the pigments to be identified and their distribution to be studied, apart from two shades of purple of organic composition. Consequently, the potential of high-pressure liquid chromatography tandem mass spectrometry (HPLC-MS/MS) was explored for the first time with application to Japanese woodblock prints. The intrinsic sensitivity of the instrument and an effective extraction protocol allowed us to identify a mixture of dayflower (Commelina communis) blue and safflower (Carthamus tinctorius) red in purple samples constituted of 2-3 single fibers. In addition to the innovative integration of MA-XRF and HPLC-MS/MS to investigate these delicate artworks, the study concluded on the use of traditional sources of colors alongside newly introduced pigments in late Edo-period Japan. This information is extremely important for understanding the printing practices, as well as for making decisions about display, conservation, and preservation of such artworks.