Salmonella enterica serovar Typhimurium (henceforth Salmonella) is a common cause of gastroenteritis in humans. Salmonella bacteria infect the intestine and actively invade intestinal epithelial cells (IECs), where they either remain in the Salmonella-containing vacuole (SCV) or escape into the cytosol. The determinants of these different life-style remain poorly understood. Research on those two distinct populations of Salmonella inside epithelial cells has mostly been done on cancer cell lines, which are known to lack components of the in vivo tissue that might be crucial to infection with Salmonella. The growing field of organoid research provides new models, such as organoid-derived monolayers, that bridge the practicability of cell lines with a higher level of complexity, by providing different cell types of a certain tissue, in a 2D monolayer structure. Here we describe and characterize a human small intestinal epithelial organoid-derived monolayer model for infection with Salmonella, using single-cell RNA sequencing (scRNA-seq). We furthermore infect the organoid-derived monolayer model with Salmonella and investigate the existence of vacuolar and cytosolic Salmonellarespectively, using a staining for the SCV surface marker LAMP-1. To study the dynamics of the infection, we have implemented a high-throughputmethod, combining single-cell RNA-seq and RNA metabolic labeling. Altogether, we expect our multi-dimensional single-cell analysis to reveal the host factors that underliethe fate decision of bacterial divergent lifestyles.