The tomato (Solanum lycopersicum) is a widely cultivated crop worldwide that serves as a model system for fruit development studies. Agrobacterium tumefaciens–mediated transformation of tomato has played a central role as a tool for analyzing the function of candidate genes and producing transgenic lines with enhanced resistance to pathogens, tolerance to abiotic stresses, and improved fruit quality traits. Among the many tomato varieties, the miniature dwarf cultivar Micro-Tom (MT) has been increasingly adopted as a model system for tomato research due to its short life cycle, small size, and high transformation efficiency. This protocol outlines a replicable methodology for A. tumefaciens–mediated transformation of Micro-Tom from cotyledon explants, utilizing cost-effective plant growth regulators for shoot regeneration, high transformation rates, reduced regeneration time, and enhanced rooting conditions.