Shock-like water stress using hydroponics and gradual water deficit in soil are the two widely used treatments to analyze transcriptional response of many crops to drought. In this study, we investigated the effects of shock drought (ShD) (0, 0.5, 1, 4, and 8 h) and slow drought (SDD) [soil water content (SWC) of 35 and 50 %] on the expression of well-known drought-responsive genes supplemented with physiological changes in barley. Two barley cultivars with contrasting leaf relative water content (RWC) and water loss rate (WLR) values were selected as Marti (MR; 60 % RWC and 0.046 gh(-1) g(-1) DW) and Erginel90 (ER; 38.3 % RWC and 0.350 gh(-1) g(-1) DW) under 38 % of SWC condition. According to the results, 0.5 h ShD was the critical time point for stress perception in leaves defined by the increase in WLR, ion leakage and H2O2 concentration. Expressions of antioxidant-related genes (Cu-Zn/SOD, HvCAT2, HvGST6, HvAPX) were rapidly induced in MR at 8 h shock, while only slightly upregulated in ER. We have also observed higher induction of expressions of HvBAS1, HvMT-2, HvABA7 and a photosynthesis-related gene HvLHCB during ShD compared to SDD. Contrarily, transcription factors (TFs), HvWRKY12 and HvDRF1 were expressed with lower values during shock-dehydration. Slow-drought treatments in both cultivars were characterized with high leaf RWCs and osmotic adjustment with low cell membrane damage, suggesting that barley maintains a basal tolerance to long-term water deficit. Our results confirmed that type of water stress treatment is crucial to measure gene expression, and a shock-like dehydration method should be the treatment of choice in evaluating barley plants with different physiological characteristics for water tolerance.