Listeria monocytogenes is a bacterial pathogen that enters and proliferates in the cytosol of mammalian cells. Following entry, bacteria disrupt the invasion vacuole and reach the cytoplasm, where they replicate and use the actin cytoskeleton to propel themselves from cell to cell [1, 2]. To achieve this cytosolic lifestyle, Listeria deploys virulence effectors that hijack diverse cellular processes [3, 4]. Human epithelial cells grown in vitro can be used to study the L.\u00a0monocytogenes infectious process. However, rapid multiplication and dissemination of bacteria may induce cell death and detachment, thereby forming lytic plaques. Thus, in vitro infections with L.\u00a0monocytogenes have been restricted to short time courses (usually from a few minutes to one day). In order to study L.\u00a0monocytogenes long-term infections, we have set up a protocol, with several modifications to the gentamicin protection assay previously used for short-term infections with this pathogen . In a subset of human cells, such as hepatocytes or trophoblast cells, this protocol enables to observe and study the entrapment of L.\u00a0monocytogenes within vacuoles, termed \u201cListeria-Containing Vacuoles\u201d (LisCVs), after the intercellular dissemination phase of Listeria.Here, this protocol describes the assays used to study LisCVs in a JEG3 trophoblast cell monolayer.