Abstract: Being one of the most important parts of polychrome cultural relics, a binder is used as an adhesive of pigments, but it is closely related to various kinds of cultural relic diseases. Recently, there are some reports on analyses of binders in artworks, such as egg, animal glues, milk (casein), plant waxes, oils, resins, polysaccharide materials and so on. However, methodological study of proteinaceous binders in artworks by gas chromatography-mass spectrometry (GC-MS) is rarely reported. In this study, a method of GC-MS was developed for identification of proteinaceous binders in artworks depending on determination of the eleven common amino acids (alanine, glycine, valine, leucine, isoleucine, serine, proline, phenylalanine, aspartic acid, glutamic acid and hydroxyproline). The mainly analytical procedure was as follows: 1) extraction by ammonia with an ultrasonic cleaner; 2) enrichment and purification by a C4 solid phase extraction column; 3) digestion to amino acids in a microwave oven with 6 mol/L HCl; 4) derivatization by using N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) with 1% tert-butyldimethylchlorosilane as a derivatization reagent; 5) analyses by a 7890A-5975C GC-MS (Agilent, USA) with norleucine as the internal standard. The results showed that they had good linear relationships with the linear correlation coefficients (R) of no less than 0.99, and the limits of detection of the amino acids ranged from 0.066 to 0.676 μg/g. The relative standard deviations (RSDs) were from 2.7% to 6.3% of 11 kinds of mixed standard solutions (n=6), and RSDs were below 5% excepting for that of serine and hydroxyproline. Based on the analyses of three kinds of modern reference samples (pigskin glue, egg and milk), the results showed that each binder was characterized by a specific set of amino acid percentages, which could be used as a “fingerprint”. Animal glue binder contains hydroxyproline (Hyp) as a special amino acid and has high percentage of glycine (Gly). Egg binder has high percentages of aspartic acid (Asp) and serine (Ser), while milk has high percentage of glutamic acid (Glu). Moreover, based on combination of principal component analysis (PCA), the proteinaceous binders in the golden wire of Guangxi clothing were identified as mixture glues consisting of animal glue and egg. From experimental results it has been proved that GC-MS is suitable for quantitative analysis of proteinaceous binders in artworks.