ABSTRACTThe molecular characterization of progressively complex paint systems in cultural heritage objects remains a significant challenge, particularly for artworks employing mixed synthetic/natural binder. Overcoming this challenge is critical for reconstructing the material history and informing conservation strategies. This study investigates the multi‐layered hybrid binder stratigraphy of Portrait of Farrokh Gaffari (Fathollah Qollar Aghasi, 1959), a representative work of the Iranian Coffee House School. Through complementary pyrolysis approaches—conventional pyrolysis‐gas chromatography‐mass spectrometry and thermally assisted hydrolysis and methylation pyrolysis‐gas chromatography‐mass spectrometry—the article developed an optimized analytical protocol for differentiating organic components in complex artistic matrices. Results demonstrate a four‐layer structure: (1) cotton canvas substrate, (2) ground layer, (3) pigment layer, and (4) surface varnish. Targeted molecular marker analysis revealed the chemical stratigraphy: (1) dibutyl phthalate‐plasticized polyvinyl acetate in the ground layer, (2) linseed oil as the binder in the pigment layer, and (3) an alkyd resin varnish incorporating phthalic acid, pentaerythritol, linseed oil derivatives, and terpenoids from Pinaceae resin. The combined pyrolysis approaches successfully addressed analytical challenges associated with microsamples from mixed‐media artworks, enabling clear differentiation among native binders, synthetic polymers, and additives. This research bridges critical gaps between analytical chemistry and heritage science: demonstrating how molecular diagnostics directly informs preventive and interventive conservation strategies for 20th‐century hybrid artworks.