In order to analyze root proteins involved in drought tolerance in poplar, a proteomic study was conducted on the roots of two Populus deltoïdes × Populus nigra cultivars, Carpaccio and Soligo. These cultivars were selected based on contrasted physiological responses under water stress. Plants were exposed to different water stress levels (Control, Early, Medium and Severe), and root tips collected. Technical optimization included protein extraction and resolubilization, as well as the finetuning of electrophoretic conditions. The best results were obtained upon using phenol extraction, a resolubilization solution containing two reduction reagents, DTT and 2-ME, and 100 μg protein load on both acidic (pH 4-7) and basic (pH 7-11NL) IPG strips. A total of 48 2-D gels were then produced and quantitatively analyzed using SameSpot Progenesis software. Statistical analyses were completed using normalized spot volumes. Multivariate analyses (correlation matrix, PCA and HCA) and twoway ANOVAs showed that protein expression was first regulated by genotype effect and second by treatment effect. Differentially regulated proteins are being identified based on tandem mass spectrometry. This functional information should shed some lights on the molecular players involved in drought-stress response and potentially underlying adaptation to this abiotic constraint.