A new two-step analytical protocol has permitted the reliable structural identification of red wine thanks to the presence of dark grape (tartaric, malic, syringic acids) and fermentation markers (succinic and pyruvic acids) in a smashed, large, coarse jar and a jug excavated inside a Neolithic house destroyed by fire around 4300 BCE at the site of Dikili Tash in northern Greece. This new method, which has also been tested successfully on other vessels, exploits the chemical break-down of the clay and the simultaneous liberation and derivatization of biomarkers. Since aldaric acids are not extracted by a simple solvent extraction, but only when submitted to the second acido-catalyzed extraction, their detection in the second extract indicates organic residues are more deeply impregnated and bound to the clay structure than previously thought. Chromatography coupled with mass spectrometry leads to the very sensitive detection (<10 ng/g sherd for tartaric acid, i.e. < 10−6 mL of wine/g sherd) and reliable identification of fermented grape biomarkers. Their identification in a Neolithic jar from Dikili Tash corroborates the finding of pressed grapes consisting of loose pips, skins, and pips still enclosed by skin in association with this jar. Our results demonstrate Neolithic wine-making in the northern Aegean, and provide the earliest solid evidence for the Eastern Mediterranean and Europe. This new method could be more widely used for detecting wine traces in all sorts of archaeological artefacts or structures. It constitutes an essential tool for a better understanding of wine-making and of contexts of consumption in ancient civilizations.