Acclimatization to stress is associated with profound changes in proteome composition. The use of plant cell and tissue culture offers a means to investigate the physiological and biochemical processes involved in the adaptation to osmotic stress. We employed a new proteomic approach to further understand the response of calli to dehydration induced by polyethylene glycol (PEG6000). Calli of three durum wheat genotypes Djenah Khetifa, Oued Zenati and Waha were treated with two concentrations of polyethylene glycol to mimic osmotic stress. Changes in protein relative abundance were analyzed using a new electrophoretic approach named diagonal two-dimensional electrophoresis (D-2DE), combined with mass spectrometry. Total proteins were extracted from 30-day-old calli from three durum wheat genotypes that showed contrasting levels of drought stress tolerance in the field. The combination of one-dimensional electrophoresis and D-2DE gave a specific imprint of the protein extracts under osmotic stress, as well as characterizing and identifying individual target proteins. Of the variously expressed proteins, three were selected (globulin, GAPDH and peroxidase) and further analyzed using qRT-PCR at the transcriptome level in order to compare the results with the proteomic data. Western blot analysis was used to further validate the differences in relative abundance pattern. The proteins identified through this technique provide new insights as to how calli respond to osmotic stress. Our method of study provides an original and relevant approach of analyzing the osmotic-responsive mechanisms at the cellular level of durum wheat with agronomic perspectives.