Drought is one of the main causes of mortality in holm oak (Quercus ilex) seedlings used in reforestation programs. Although this species shows high adaptability to the extreme climate conditions prevailing in Southern Spain, its intrinsic genetic variability may play a role in the differential response of some populations and individuals. The aim of this work was to identify proteins and derived proteotypic peptides potentially useful as putative markers for drought tolerance in holm oak by using a targeted post-acquisition proteomics approach. For this purpose, we used a set of proteins identified by shotgun (LC-MSMS) analysis in a drought experiment on Q. ilex seedlings from four different provenances (viz. the Andalusian provinces Granada, Huelva, Cadiz and Seville). A double strategy involving the quantification of proteins and target peptides by shotgun analysis and post-acquisition data analysis based on proteotypic peptides was used. To this end, an initial list of proteotypic peptides from proteins highly represented under drought conditions was compiled that was used in combination with the raw files from the shotgun experiment to quantify the relative abundance of the fragment’s ion peaks with the software Skyline. The most abundant peptides under drought conditions in at least two populations were selected as putative markers of drought tolerance. A total of 30 proteins and 46 derived peptides belonging to the redox, stress-related, synthesis,-folding and degradation, and primary and secondary metabolism functional groups were thus identified. Two proteins (viz., subtilisin and chaperone GrpE protein) were found at increased levels in three populations, which make them especially interesting for validation drought tolerance markers in subsequent experiments.