Screening plant genetic resources under water deficit environment using reliable selection criteria is an essential step in breeding programs towards drought tolerance improvement. In this greenhouse experiment, the twenty-five barley (Hordeum vulgare L.) cultivars were subjected to well-watered and water deficit stress conditions in order to measure changes of antioxidant enzymes activities, malondialdehyde (MDA), proline and protein contents in response to water deficit stress and to evaluate the significance of the biochemical traits as selection criteria for improvement of grain yield. Significant variations were observed among the barley cultivars for grain yield as well as the biochemical traits including the antioxidant enzymes, MDA, protein and proline contents. Water deficit stress caused a significant increase in MDA, proline and protein contents as well as the antioxidant enzymes activities including catalase, peroxidase, superoxide dismutase and ascorbic peroxidase. The results of correlation coefficients and regression modeling showed that the relationships between grain yield and biochemical traits were somewhat influenced by water regimes. The results suggested that high proline content along with low protein content can be used as the criteria for selection of high-yield barley cultivars under well-watered conditions. Catalase and superoxide dismutase activities as well as proline content were known as the selection criteria with significant contributions to grain yield of barley under water deficit conditions. Catalase and superoxide dismutase activities were significantly positively related to grain yield, while proline content had a negative relationship with grain yield under water deficit conditions.