SummaryLinks between bacterial infections and cancer are actively investigated. Cost-effective assays to quantify bacterial oncogenic potential can shed new light on these links. Here, we present a soft agar colony formation assay to quantify mouse embryonic fibroblast transformation afterSalmonellaTyphimurium infection. We describe how to infect and seed cells in soft agar for anchorage-independent growth, a hallmark of cell transformation. We further detail automated cell colony enumeration. This protocol is adaptable to other bacteria or host cells.For complete details on the use and execution of this protocol, please refer to Van Elsland et al.1