Holm oak (Quercus ilex) is the most important and representative specie of the Mediterranean forest and of the Spanish agrosilvo-pastoral ecosystem “Dehesa”. Despite its environmental and economic interest, Holm oak is still an orphan species whose biology is very little known, especially at the molecular level. In this research, we have performed a shotgun proteomic approach (nLC-MSMS, Orbitrap) to analyze the Holm oak proteome, using, as starting material, a pool generated by mixing equal amounts of homogenized tissue, including embryo, cotyledons (from mature acorns), and leaves and roots (from 6-month old plantlets grown in a greenhouse under environmental conditions). The proteome generated will be the bases of further studies on population variability, growth, development and responses to stresses in this species.