In studies of the materials in old master paintings, the characterization of thin and degraded layers often presents unusual challenges for routinely used methods or analysis. This paper discusses analyses performed as part of a broader study on the materials and techniques of Pontormo's Portrait of Alessandro de' Medici (1534-1535). Pontormo built up the portrait using a complex sequence of preparation, drawing and paint layers, the analysis of which was further complicated by the presence of degraded materials in some areas of the paint surface. Characterization of the multiple layers used by Pontormo in this portrait and of the degraded surface material was carried out using gas chromatography/mass spectrometry (GCMS), Fourier transform infrared microspectroscopy (FTIR) and scanning electron microscopy with energy dispersive spectroscopy (SEM-EDS). The GCMS analysis of the protein component in samples of the preparatory layers indicated the use of an egg-based medium for extensive brown undermodelling prior to the final layers in oil-bound pigments. The focus of this paper is on the approach taken in characterising the preparation layers and degraded surface materials. -- ICCROM