Drought is considered one of the main causes of mortality of holm oak (Quercus ilex) upon seedling plantations in reforestation programs. Although Q. ilex shows high adaptability to extreme climate conditions prevailing in Southern Spain, the genetic variability inherent to this species could be associated with a differential response depending on populations or individuals. The objective of this work was to identify proteins and derived proteotypic peptides to be used as markers of tolerance to drought in holm oak using a targeted post-acquisition proteomics approach. The study was based on a set of proteins identified by shotgun (LC-MSMS) from a drought experiment carried out in Q. ilex seedlings from four Andalusian provenances (Granada, Huelva, Cadiz and Seville). A double strategy, shotgun protein quantification and target peptides quantification post-acquisition data analysis based on proteotypic peptides, was used. For this purpose, an initial list of target peptides consisting in proteotypic peptides derived of proteins highly represented in drought conditions was compiled. Then, the proteotypic peptides list and the raw files from the shotgun experiment were imported into the Skyline software to quantify the relative abundance of the fragments ion peaks. Those peptides significantly more abundant in drought conditions at least in two populations were selected as putative markers of tolerance to drought. These corresponded to proteins belonged to redox, stress related proteins, synthesis, folding and degradation, and primary and secondary metabolism functional groups. Two proteins were found to be increased in three populations, making them especially interesting for validation in subsequent experiments as markers of drought tolerance.