The mosaic in the apse of the Church of S. Agnese fuori le mura in Rome represents one of the most important examples of the wall mosaics of the Roman Middle Ages. Although it is associated with Byzantine figurative culture, no scientific study has addressed this important piece of Italian art history. One factor that has probably limited and created difficulties for its analysis is the poor legibility of the original parts, which are compromised by heavy restorations affecting the gold background, the faces of the two male side figures, and the lower band of the mosaic. The present work describes how multispectral imaging provided significant guidance in the preliminary identification of possible original areas in this ancient wall mosaic. Through an interdisciplinary approach, the art historical background and historical graphic documentation of known restorations supported the use of multispectral imaging to recognize original parts. The initial results of the lab analyses (SEM-EDS and EMPA) of supposed original tesserae validated the hypothesis made a priori thanks to multispectral acquisition, opening up new application possibilities for use of this noninvasive technique in the preliminary in situ identification of original parts in restored glass wall mosaics.