Fires can have a negative impact on the environment, human health, property and ultimately also on various objects of cultural heritage (CH). This paper deals with an investigation into the degradation of selected proteinaceous paint layers that were exposed to fire-related effects (i.e., fire effluents and/or high temperatures) in a modified cone-calorimeter system. Paint layers of egg yolk adhesive (E) and lead white tempera (E + LW) were exposed to fire-related impacts on top of a CH stack and in a specially designed CH test chamber. On the CH stack, the proteinaceous paint layers were exposed to fire effluents and high temperatures, while in the CH test chamber, the samples were exposed mainly to fire effluents. The molecular changes to the exposed paint layers were analysed by invasive and non-invasive spectroscopic analyses (i.e., FTIR and Raman spectroscopy) and complimented with pyrolysis-GC-MS, while the colour changes were evaluated using colourimetry. It was concluded that the proteinaceous binder degrades into aromatic amino acids and/or fatty acids after exposure to the overall impacts of the fire. Aromatic amino acids were detected by means of the FTIR and py-GC-MS analyses. In the case of the lead white tempera exposure, partial dissociation of the lead white pigment was confirmed by the detection of alteration products, such as lead oxide and lead carbonate. Moreover, the investigation of the E + LW samples exposed for longer times revealed the presence of lead carboxylates. On the other hand, no significant molecular changes were observed with the CH samples exposed to fire effluents in the CH test chamber. The research offered us an insight into the fire-induced effects on selected paints for the first time.