Introduction: Tomato (Solanum lycopersicum L.) is one of the essential vegetables worldwide, for consumption and mitigating malnutrition. Genetic transformation conceivably overcomes its yield challenges due to salinity, a crucial constraint for the economical use of 30% of cultivable lands in the coastal region of Bangladesh. Therefore, a robust and reproducible protocol has been established for in vitro regeneration and transformation to develop transgenic salt-tolerant tomato plants. Materials and Methods: During micropropagation, cotyledonary leaf explants were excised and cultured on MS media containing different combinations and concentrations of plant growth regulators. In transformation, the pre-cultured explants were inoculated and co-cultivated with Agrobacterium. Then they were transferred to the antibiotics-supplemented media to achieve salt-tolerant putative transformed plants. The transformation was confirmed by P-glucuronidase (GUS) assay and PCR for the antiporter gene. Results: Maximum regeneration response was achieved from the explants abaxially positioned at a 1.5 cm distance apart. BARI Tomato 14 and BINA Tomato 3 showed the highest shoot regeneration response (%) on MS media supplemented with 2 mg/L BAP and with 0.1 mg/L IAA for BARI Tomato 2 and BARI Tomato 15. Bacterial culture of OD600 0.68 for 30 min and a Co-cultivation period of 48 h resulted in the highest transformation frequency (47%) in Agrobacterium-mediated transformation with pBI 121 in BARI Tomato 3. The highest regeneration frequency (20.5%) was obtained in transformation with pH7WG2_OsNHX1_1.6. Conclusions: The optimized procedure is simple, efficient, and can be used for micro-propagation and the production of tolerant varieties to increase yield in saline areas.