This study aims to investigate the activities and expression of enzymes of primary metabolism and relate these data with the growth performance of three different durum wheat genotypes (Maali; YT13; and ON66) under osmotic stress. Growth traits—including plant height, dry weight (DW) and relative water content (RWC)—were measured to classify genotypes depending on their tolerance to stress. Several enzymes were investigated: Ascorbate peroxidase (APX), Glutamine Synthetase (GS), Glutamine dehydrogenase (GDH), Glutamate synthase (GOGAT), Glucose 6-phosphate dehydrogenase (G6PDH), and Phosphoenolpyruvate Carboxylase (PEPC). The expression of the cytosolic and plastidic glutamine synthetase (TaGS1 and TaGS2), high affinity nitrate transporters (TaNRT2.3) and Glutamate dehydrogenase (TaGDH) were also detected by qRT-PCR. The results indicated different growth performances among genotypes, indicating Maali and YT13 as tolerant genotypes and ON66 as a drought-susceptible variety. Data showed a decrease in PEPC and increase in APX activities under osmotic stress; a slight decrease in GS activity was observed, together with an increase in G6PDH in all genotypes; GS and NRT2 expressions changed in a similar pattern in the different genotypes. Interestingly, Maali and YT13 showed higher transcript abundance for GDH under stress compared to ON66, suggesting the implication of GDH in protective phenomena upon osmotic stress.