The investigation was carried out to evaluate the influence of glutathione (GSH: levels 0, 0.1, 0.5 1.0, and 2.0 mM) on the callus growth, shoot multiplication and phytochemicals of in vitro shoots of the Date palm cv. Barhee. The optimum concentration of GSH was 1.0 mM. Such concentration improved the callus growth and increased its weight to 312.0 mg, the addition of this substance of the same concentration level showed the highest response rate and the number of shoots per jar (73.34 ± 2.69 and 8.83 ± 0.80 shoots/jar, respectively). Also, the 1.0 GSH application resulted in reducing the percentage of browning to 0.0 compared to the other concentrations. The results of the biochemical analysis revealed that treatments GSH of 0.1, 0.5 and 1.0 mM reduced the total soluble phenols compound (TSPC). These treatments were also more effectively reducing peroxidase (POD) and phenylalanine ammonia-lyase (PAL) activity than the concentrations of 2 mM or 0 (control treatment).