An innovative GC/MS procedure for the characterization of organic materials in samples from works of art was developed. It is based on a multistep chemical pretreatment of the samples based on the ammonia extraction of proteins and polysaccharide materials, in order to separate them from lipid and resinous materials. The extraction is then followed by the separation and purification of proteinaceous and polysaccharide materials before hydrolysis, based on the use of monolithic sorbent tip technology with a C4 stationary phase. Lipids and resins are saponified/salified separately. Three fractions are generated and analyzed separately by GC/MS, thus enabling a quantitative analysis to be performed on aldoses and uronic acids, amino acids, mono- and dicarboxylic aliphatic acids, to determine polysaccharide, proteinaceous, and glycerolipid materials and molecular pattern recognition for the natural resin and wax components. With this analytical procedure, for the first time, glycerolipids, natural waxes, and proteinaceous, resinous, and polysaccharide materials can be simultaneously characterized in the same microsample from painted works of art. This new analytical approach prevents any analytical difficulties arising when the sample is divided into several different aliquots to be chemically processed separately, in order to characterize the various classes of organic materials. The procedure was successfully applied to samples from paintings from the Bamiyan Buddhas and a panel painting from the 15th century, highlighting the occurrence of glycerolipids, animal and plant resins, proteinaceous and polysaccharide materials.