Salmonella enterica in agricultural environments has become an important concern, due to its potential transmission to humans and the associated public health risks. To identify genes contributing to Salmonella adaptation to such environments, transposon sequencing has been used in recent years. However, isolating Salmonella from atypical hosts, such as plant leaves, can pose technical challenges due to low bacterial content and the difficulty to separate an adequate number of bacteria from host tissues. In this study, we describe a modified methodology using a combination of sonication and filtration to recover S. enterica cells from lettuce leaves. We successfully recovered over a total of 3.5 × 10 6 Salmonella cells in each biological replicate from two six-week old lettuce leaves, 7 days after infiltration with a Salmonella suspension of 5 × 10 7 colony forming units (CFU)/mL. Moreover, we have developed a dialysis membrane system as an alternative method for recovering bacteria from culture medium, mimicking a natural environment. Inoculating 10 7 CFU/mL of Salmonella into the media based on plant (lettuce and tomato) leaf and diluvial sand soil, a final concentration of 10 9.5 and 10 8.5 CFU/mL was obtained, respectively. One millilitre of the bacterial suspension after 24 h incubation at 28 • C using 60 rpm agitation was pelleted, corresponding to 10 9.5 and 10 8.5 cells from leaf-or soil-based media. The recovered bacterial population, from both lettuce leaves and environment-mimicking media, can adequately cover a presumptive library density of 10 6 mutants. In conclusion, this protocol provides an effective method to recover a Salmonella transposon sequencing library from in planta and in vitro systems. We expect this novel technique to foster the study of Salmonella in atypical hosts and environments, as well as other comparable scenarios.