Soybean (Glycine max L.) is an important cash crop. It is a source of protein and vegetable oil. In Pakistan, it is subjected to various abiotic stresses such as; salt stress, which are globally a major threat to agriculture, and which decline crop production resulting in food insecurity. To overcome these challenges, the development of salt-tolerant varieties helps to increase crop yield in those areas which are unfit for cultivation due to high salinity. The present work was established to evaluate the suitability of tissue culture, which explores the genetic potential of tissue culture cell lines for salt-tolerant varieties. Cotyledonary callus of explants was germinated on MS media at different NaCl concentrations (0, 50, 100, 150 and 200 mM). Salt tolerance was analyzed on morphological (callus, roots and shoots number and length), physiological (Membrane thermo stability (EC), lipid peroxidation (MDA) and hydrogen peroxide (H2O2) and biochemical (proline content, total soluble protein and SDS-PAGE) markers. Our results revealed that high salt concentration affects the weight of callus, weight, length and number of shoots and roots. It also increased the physiological parameters, like, EC, MDA and H2O2. Biochemical parameters, such as protein and proline content, were higher in salt-treated plants as compared to non-treated plants. SDS-PAGE also showed different expressions of proteins under salt stress conditions. The results indicate the potential of the plant tissue culture method as successful and consistent for carrying out salt tolerance studies in Glycine max. The method is easy to control, so the tolerance capability of the plants can be well determined. Tissue culture is a reliable method for producing salt-tolerant cell lines. In future, other methods like genetic engineering and plant breeding will be essential for producing salt-tolerant crop varieties along with tissue culture methods.