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The scientific investigation of the techniques employed by the artist, such as composition of the paints, color palette, and painting style represents a fundamental pre-requisite in order to develop proper conservation and restoration strategies. In this context, the combined use of non-destructive, non-invasive in situ image and chemical analyses was here successfully employed for the investigation of the XVIII century Madonna della Lettera panel painting from the ancient Basilian abbey of Santa Maria di Bordonaro in Messina (Italy). The used techniques were visible, infrared (IR) and false-color infrared (FCIR) photography, X-ray radiography, X-ray fluorescence (XRF) and Raman spectroscopy. The goal was to obtain accurate information on materials and techniques originally used and possible later interventions, with particular regard to the nature of the painting materials. From the results, details of the artwork useful for restoration and conservation procedures were revealed. The identification of most of the artist's palette was also achieved: Prussian blue for blue color, lead white for white, umber for the brown, cinnabar for the red, and carbon black for the black color. The composition of different preparatory substrates was also investigated. The obtained results, other than constituting a crucial step for future restoration works, can be at the same time useful for the dating of the painting, that does not report the date and the artist's signature.