The paper presents the results of the multi-analytical study of the painting on a panel from the icon collection of the Gallery of Matica srpska museum in Novi Sad, Serbia. It is part of the research aiming to set the methodology for the museum’s database on artistic materials and techniques present in the collection. Computer tomography (CT) scanning was used to understand the structure of the wooden panel support. Ultraviolet (UV) and infrared (IC) imaging, as well as visible (VIS) macro photography, were used to study the paint layer, both the original part and restoration treatments, as well as the coat of varnish. Energy dispersive X-ray fluorescence (EDXRF) and Fourier-transform infrared (FTIR) spectroscopy revealed the pigments, binders, and metal leaf, defining the artistic technique. Optical microscopy (OM) and scanning electron microscopy (SEM) with energy-dispersive X-ray spectroscopy (EDS) were used to disclose the stratigraphy and composition of layers in the artwork. The multi-analytical approach confirmed that protein-based binder, gilding, silver leaf, and traditional pigments were used. The data gathered from this research are important for studying the artistic materials and techniques in icon production and defining the methodology setting for the museum collection’s databases as the reference material.