Lettuce (Lactuca sativa L.) plays a pivotal role in global agriculture and food supply chains as a widely cultivated commercial crop. This chapter presents a detailed protocol for transforming lettuce using Agrobacterium tumefaciens. The procedure includes a two-day pre-culture period of cotyledon explants, followed by a 20-min inoculation with Agrobacterium without explant wounding, and a two-day co-culture on the same medium. Afterward, the selection and regeneration phases under kanamycin selection led to the establishment of transgenic plants in the greenhouse. The entire process from the initial explant infection to the greenhouse cultivation of transgenic plants spans roughly 120 days, achieving an average transformation efficiency of 45%. This protocol offers a reliable method for the genetic modification of lettuce, for use in research or crop improvement.