Background: In this study, lettuce roots and leaves were contaminated with enterohemorrhagic Escherichia coli O157:H − strain 431/97 under greenhouse conditions. Furthermore, the internalization of strain 431/97 in lettuce roots and leaves was examined. To track the inoculated bacteria during the experiments and to differentiate them from the autochthonous microbiota, a combined protocol including matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and PCR was established. Results: At different time points after inoculation of Batavia lettuce with 5.5 × 10 6 cfu/mL (high inoculation level) or 3.0 × 10 1 cfu/mL (low inoculation level) E. coli O157:H − strain 431/97 as well as sterile 0.9% (w/v) sodium chloride solution (negative control), samples from the root and the leaf were taken and surface disinfected with gentamicin. After homogenization, microorganisms were isolated from the samples and analyzed by MALDI-TOF MS. Analysis of the root samples resulted in bacterial counts of 1.0 × 10 2-1.0 × 10 6 cfu/0.25 g depending on the inoculated viable counts and the incubation period. In the leaf samples, strain 431/97 was not detected. The investigation of the viable cell counts of E. coli O157:H − 431/97 following irrigation of the leaves resulted in bacterial counts of 10 2 cfu/0.25 g for the disinfected leaf samples. Conclusions: Thus, the established protocol is suitable for detecting the investigated strain under greenhouse conditions in plant infection experiments. This strain may indeed survive in the soil, but did not enter the plant via the root in detectable numbers. Contrarily, viable counts exceeding the generally accepted infective dose of less than 100 cells for enterohemorrhagic E. coli were determined internalized after irrigation of the leaves. As this may pose a risk for the consumer, the present study provides a valuable set of tools for further research.