Soybean is a widely cultivated oilseed and high protein source crop around the globe. Due to their high demand, soybeans are attractive to researchers in genetic engineering. However, soybean is a recalcitrant crop with low transformation efficiency and offers no genotype flexibility. Agrobacterium tumefaciens–mediated and particle bombardment methods of gene transformation are the most practiced transformation methods in soybean. The robust strains of Agrobacterium like GV3101 or EHA105 are preferred for soybean transformation. Transformation efficiency is not only dependent on the genotype of soybean but also on the choice of explant. Cotyledonary node and half seed are commonly used explants for soybean transformation. Here, we describe the Agrobacterium-mediated genetic transformation of soybean cultivars using the GV3101 strain through a split cotyledonary explant. This protocol is optimized for local soybean varieties (i.e., Faisal, Super) using GUS and GFP as reporter genes.