Soil salinization is an abiotic stress factor seriously limiting agricultural production. This study aimed to investigate the impact of salt stress on barley seedlings (Hordeum vulgare L.) and to determine which salt stress indicators are primarily activated as tolerance mechanisms in the early stages of seedling development. Seven-day-old plants, grown under controlled conditions of the growth chamber, were exposed to different sodium chloride concentrations (50, 100, 200, 400 mM). The effect of salt stress was monitored after 3, 6, 12, and 24 hours. Physiological and biochemical measurements were performed on the first fully developed leaves and roots of barley seedlings. As stress levels increased, proline content and relative water content (RWC) also increased, indicating the importance of osmotic adaptation of seedlings and their resistance to possible dehydration due to osmotic stress. Decreased malondialdehyde levels showed a decrease in oxidative damage of cellular lipids, which is a feature of stress-tolerant plants. Sensitivity to damage in salt stress conditions still exists as indicated by decreased levels of photosynthetic pigments: chlorophyll a, chlorophyll b, and carotenoids. Chlorophyll a fluorescence parameters (L- and K-band; vitality index - PIABS) confirmed very good functionality of the photosynthetic apparatus of barley seedlings after short-term salt stress induction. This research could contribute to understanding the mechanisms of the osmotic component of salt stress. The results could also be useful for developing tools to improve the tolerance of commercially important cereals to maintain yield stability under increased salinity.