This work regards the study of painting techniques in Portuguese workshops of the 15th and early 16th centuries, specifically addressing the methodology used on the preparation of ground layers. The influence of Flemish painting in Portugal is evident in stylistic and iconographic themes of that period. As regards the painting materials, we confirmed that this influence also extended to the ground layer technique. The use of a sizing layer with calcium sulphate or garlic to isolate the support from the calcium carbonate layer was verified by SEM-EDS but not confirmed by μ-XRD or μ-Raman spectroscopy. This work is part of a larger project, “The invisible ground layer and its influence in Portuguese paintings from the 15th and 16th centuries: a question to be settled”.