Holy icons created in the Byzantine era are a vital entity in Orthodox Christianity, a living tradition unbroken over more than 1500 years. The importance of these symbolic representations has inspired interdisciplinary studies to better understand the materials and process of their construction. Researchers from a variety of fields are working together to place icons in their proper historical and cultural framework, as well as to develop long-term conservation strategies. In this Account, we review very recent analytical results of the materials and painting methods used in the production of Byzantine iconography. The care of icons requires particular attention because of their function; icons are objects of veneration and, for the most part, still stand in today's churches to serve ritual practices. Accordingly, they are affected by random, fluctuating environmental conditions aggravated by public access. Because of the holiness of the icons, the typical tradition of the church is to periodically restore the depicted scenes, either by retouching any defects or by partial or complete overpainting. These interventions greatly increase the complexity of the paint stratigraphy. To reveal the extent and quality of the original painting under several historical overpaintings or dirt overlays on the icon, researchers usually pursue a manifold approach, combining complementary multispectral imaging and spectroscopic techniques nondestructively. Unfortunately, a visual and exhaustive spectroscopic examination of a minimum number of cross-sectional microsamples is almost always necessary to clarify the structure of the paint layers and map the constituent materials identified therein. A full understanding of these details is critical for assessing the painting methods, stylistic conventions, and compositional concepts that render the different iconographic details. Cross-sectional micro-Raman spectroscopy, although time-consuming, now affords the direct identification of the distinct grains of almost all of the inorganic pigments and inert components included in the paint layers. Micro-Raman studies are complemented and cross-checked by micro-FTIR and scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS) studies. This approach is essential in documenting the evolution of the materials and techniques used in creating icons over the centuries. Analytical data on Greek icons are now available for comparison with similar results from other important schools of iconography, such as in the eastern Mediterranean, the Balkans, or Russia, or, further, with Western schools of painting. The research constitutes a reference base for identifying and solving analytical problems, such as those related to the organic materials found in icons that have not yet been systematically studied. Moreover, the results on icons are also generally applicable to important analytical issues encountered in studying any multilayered paint stratigraphies.