Compared with gas chromatography-mass spectroscopy (GC-MS) and high performance liquid chromatography (HPLC), attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy is an “analysis-without-separation” technique so that the analytical procedures can be simplified and the consumption of the art samples can be minimized in the analysis of color layers in paintings. This study examined the feasibility of the ATR-FTIR spectroscopy for identifying the protein binders in color layers. The fresh and accelerated UV light aged reference samples with single protein binders and the mixtures of pigment-protein binders were prepared, by using milk, animal glue, egg white as protein binders, and chalk, azurite as pigments. Second derivative infrared (SD-IR) spectral pattern recognition models were obtained by using principal component analysis (PCA). The protein binders in artificial art samples can be discriminated by the pattern recognition models, which were established based on the analysis of fresh samples. Therefore, the ATR-FTIR spectroscopy has great potential in micro-destructive and rapid chemical identification of protein binders in the field of cultural heritage.