Drought is an abiotic stress that significantly threatens global food security by reducing crop yields. This study aimed to evaluate the drought tolerance of barley (Hordeum vulgare L.) using polyethylene glycol 6000. A hydroponic experiment was conducted to assess twenty-four barley genotypes with potential drought resilience during the seedling stage. These genotypes were subjected to four levels of drought stress, applied using PEG-6000 at concentrations of 0%, 5%, 10%, and 20%. The experiment followed a randomized factorial design with two replications. Two-way ANOVA revealed significant effects of genotype (p < 0.001) and PEG-induced drought stress levels (p < 0.001) on most measured traits, except for root number, shoot dry weight, and root dry weight. The interaction between genotype and stress level was also significant (p < 0.001), except for shoot length, root number, SPAD readings, shoot dry weight, and shoot water content. Four barley genotypes—G16, G24, G13, and G17—exhibited the highest drought tolerance. Overall, as the PEG concentrations increased, there was a decline in germination percentage, vigour index, root and shoot length, and both new and dry weight. The identified drought-tolerant genotypes show promise for cultivation in water-limited environments, as they can maintain better growth performance under drought stress. In the future, efforts should focus on field validation, genetic and molecular research, breeding programs, and collaborative initiatives to enhance drought resilience strategies under real-world conditions.