In this article, a multidisciplinary methodological approach for studying a wooden panel painting is applied. The theoretical framework, within which this research has arisen, is the application of state-of-the-art non-destructive techniques for addressing issues concerning the constituting parts and composing materials of the artwork. Hereby, a post-Byzantine icon was studied, which was dated back to 1836. It is a painting executed on a wooden panel, with a decorated wooden frame attached. The artifact was thoroughly investigated through the application of infrared thermography (IRT), multispectral imaging (MSI), and macroscopic X-ray fluorescence spectrometry (MA-XRF). These analyses provided crucial information about the verso of the painting (i.e., the wooden panel and the frame) and allowed for the revelation of important details of the recto of the painting, which were not visible due to the presence of an old, decayed varnish. Additionally, through the detailed mapping of the distribution of various chemical elements on the recto of the painting and the frame, it was possible to identify the materials used and techniques employed. It is therefore shown that, when combined, the non-destructive methodologies in consideration can provide adequate information referring to the materiality and state of preservation of panel paintings, permitting the conservator to proceed to a tailored conservation treatment.