The present study discusses new research on the analysis of a portrait of a male figure discovered under the painting of Ecce Homo, attributed to the studio of Titian (ca. 1488Titian (ca. -1576, with an estimated date in the 1550s. The portrait was examined with non-invasive methods: X-ray radiography (XRR), digital microscopy and micro-XRF. The examination of XRR images exposed the details of the painting's underlying depiction, which according to the details preserved appears to be a completed or nearly finished portrait of a standing man. The application of digital microscopy on the Ecce Homo painting's cracks enabled the identification of the work's stratigraphy. Micro-XRF performed on selected spots allowed to identify lead white, vermilion, red iron oxide, umber, carbon black and copper green in the underlying portrait. The described investigation methodology was guided by the close visual analysis of the Ecce Homo work and proved to be effective in the identification of the pigments of the hidden painting and the reconstruction of its colour palette.