In this work, we studied protein binders in micro-samples from mural paintings from two 18th century Colonial Andean churches located in the northernmost region of Chile using a proteomic approach. The protein fraction of the samples was extracted with ammonia following a previously optimized methodology and analyzed by LC–MSMS. We identified collagen as well as egg white and yolk proteins, in accordance with a tempera painting technique. In addition, we detected for the first time the presence of muscle proteins in two of the micro-samples. The collagen and muscle proteins could be ascribed to bovine (Bos taurus) and llama (Vicugna pacos), a domesticated camelid used as food source and for transportation of goods in the Andes since pre-Hispanic times. These findings provide new aspects on the knowledge of old animal glues composition and painting practices in the Andean region with reliable information for restoration and conservation work.