Spinach (Spinacia oleracea L.) is a vegetable plant with high nutritional properties. In the present work, we studied responses of in vitro shoot cultures to salt stress (0 (control), 100, 200 and 300 mM NaCl) and salt stress-induced accumulation of 20-hydroxyecdysone (20E). Our results revealed that effect of low to moderate level of salinity stress (100–200 mM) was less pronounced on growth and tissue water content (TWC) of shoot cultures compared to higher salinity level (300 mM). The salt treated shoot cultures showed better osmotic adjustment in terms of significant accumulation of compatible solutes and total soluble sugars and also higher antioxidant enzyme activity. As the NaCl stress was increased, there was a corresponding linear raise in the Na⁺ accumulation while the contents of both K⁺ and Ca²⁺ decreased significantly. We also studied salt-stress induced accumulation of a bioactive compound; 20E and results showed that 200 mM salt treated shoot cultures accumulated significantly 2.9 fold higher 20E as compared to untreated shoot cultures. The results suggest that Spinacia oleracea exhibits considerable salt tolerance with better osmotic adjustment and can be considered a suitable candidate for the production of bioactive secondary metabolite.