The response of selected physiological parameters to water deficit was investigated in three grapevine cultivars (Vitis vinifera L.): ‘Blue Portugal’, ‘Müller Thurgau’, and ‘Sauvignon Blanc’. The aim of the greenhouse experiment was to evaluate genotype-specific responses to reduced water availability and to assess the effects of water deficit on gas exchange, pigment content, chlorophyll fluorescence, and leaf water potential. Grapevine plants were grown in pots filled with perlite and subjected to five irrigation treatments ranging from 15 to 120 mL per container, applied to create a gradient of water availability. Measurements were performed over a one-month period at regular weekly intervals. Reduced irrigation generally resulted in decreased physiological performance compared to control plants. ‘Müller Thurgau’ showed the strongest reduction in chlorophyll content and gas exchange parameters under low irrigation, indicating high sensitivity to water deficit. In contrast, ‘Blue Portugal’ exhibited relatively stable pigment content under reduced water availability. Differences among cultivars in leaf water potential and gas exchange suggest contrasting water-use strategies. Overall, the results indicate higher drought tolerance in ‘Sauvignon Blanc’, while ‘Müller Thurgau’ appears to be the most sensitive cultivar.