We report the development of an indirect ELISA procedure for specific identification of chicken-egg yolk and animal glues in painting micro-samples. The results presented integrate previously published work on ELISA recognition of bovine β-casein and chicken ovalbumin in painting materials. The integrated final ELISA procedure-optimised for protein extraction, immuno-reagent concentrations, blocking solution, incubation time, and temperature-enables multiplex identification, in single samples, of proteinaceous materials, i.e. chicken-egg yolk and albumen, animal glues, and bovine milk and/or casein, mainly used by painters in the past. The procedure has been systematically tested on laboratory models of mural and easel paintings, both naturally and artificially aged, to assess possible inhibitory effects on the immuno-reaction caused by inorganic painting materials (pigments and substrates) and by protein degradation resulting from aging processes. Real samples from case studies, which had previously been investigated and characterised by spectroscopy and chromatography, were successfully studied by use of the developed ELISA procedure. The commercial availability of all the immuno-reagents used, the affordable analytical equipment, and the specificity, sensitivity, and rapidity of ELISA make this method very attractive to diagnostic laboratories in the field of cultural heritage science. Possible further developments to the analytical potential of this technique include improvement of antibody performance and inclusion of other classes of bio-molecules as analytical targets.