Two methods of analysis of organic components of colour layers of art works have been tested: IR microspectroscopy of indigo, Cu-phthalocyanine, and Prussian blue, and MALDI-TOF-MS of proteinaceous binders and a protein-containing red dye. The IR spectra distortion common for smooth outer surfaces and polished cross sections of colour layer of art works is suppressed by reflectance measurement of microtome slices. The detection limit of the three blue pigments examined is approximately 0.3 wt% in reference colour layers in linseed oil binder with calcite as extender and lead white as a drying agent. The sensitivity has been sufficient to identify Prussian blue in repaints on a Gothic painting. MALDI-TOF-MS has been used to identify proteinaceous binders in two historical paintings, namely isinglass (fish glue) and rabbit glue. MALDI-TOF-MS has also been proposed for identification of an insect red dye, cochineal carmine, according to its specific protein component. The enzymatic cleavage with trypsin before MALDI-TOF-MS seems to be a very gentle and specific way of dissolution of the colour layers highly polymerised due to very long aging of old, e.g. medieval, samples.