Activation of γ-aminobutyric acid (GABA) shunt pathway and upregulation of dehydrins are involved in metabolic homeostasis and protective mechanisms against drought stress. Seed germination percentage, seedling growth, levels of GABA, alanine, glutamate, malondialdehyde (MDA), and the expression of glutamate decarboxylase (GAD ) and dehydrin (dhn and wcor ) genes were examined in post-germination and seedlings of four durum wheat (Triticum durum L.) cultivars in response to water holding capacity levels (80%, 50%, and 20%). Data showed a significant decrease in seed germination percentage, seedling length, fresh and dry weight, and water content as water holding capacity level was decreased. Levels of GABA, alanine, glutamate, and MDA were significantly increased with a negative correlation in post-germination and seedling stages as water holding capacity level was decreased. Prolonged exposure to drought stress increased the GAD expression that activated GABA shunt pathway especially at seedlings growth stage to maintain carbon/nitrogen balance, amino acids and carbohydrates metabolism, and plant growth regulation under drought stress. The mRNA transcripts of dhn and wcor significantly increased as water availability decreased in all wheat cultivars during the post-germination stage presumably to enhance plant tolerance to drought stress by cell membrane protection, cryoprotection of enzymes, and prevention of reactive oxygen species (ROS) accumulation. This study showed that the four durum wheat cultivars responded differently to drought stress especially during the seedling growth stage which might be connected with ROS scavenging systems and the activation of antioxidant enzymes that were associated with activation of GABA shunt pathway and the production of GABA in durum seedlings.