According to treatises on 15th and 16th century paintings, artists dedicated particular attention to the sizing layer-consisting mainly of animal glue applied onto the wood support before further application of the ground layer. The stability of a painting mainly depends on the presence of a very cohesive sizing layer. However, the study of these layers has not received special attention from researchers. In this article we present a methodology for characterization of the sizing layer both chemically, by IR spectroscopy (Fourier transform infrared spectroscopy), and morphologically, by scanning electron microscopy (SEM). Secondary electron images obtained by SEM allow precise characterization of such layers. Painting reconstructions were used as references in development of the method to study the sizing layer in real painting samples. Presented herein are examples of this study on 15th and 16th century Portuguese paintings, particularly on the Triptych of S. Simão, from the Aveiro Museum, and S. Pedro, belonging to the Mercy of Tavira.