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The study of ancient works of art is a very challenging task, mainly due to the impossibility of applying experimental approaches that could damage anyhow the object from which analytical information has to be obtained. Spatially resolved analytical methods have significantly enhanced our capacity to study ancient material since they cause minimal and at times no damage to the studied object. Unfortunately, only a few analytical techniques operating within the requested spatial resolution are applicable for the investigation of the organic components of artistic and archaeological objects.In this work, iron-gall ink was prepared according to ancient recipes and its composition was evaluated by AP/MALDI-MS. The latter is demonstrated to be a very valuable tool for the study of the organic components of ancient works of art as it combines the advantages of the vacuum MALDI-MS analytical approach to the possibility of analysing samples in air. In situ analyses were also carried out by analysing strokes of ink directly from paper and parchment.