The data here reported introduce the wound‐healing assay as a tool for testing probiotics aimed at protecting gastrointestinal mucosal surfaces and to verify the consistency of their manufacturing. At the scope, we compared the in vitro effects of two multi‐strain high concentration formulations both commercialized under the same brand VSL#3 but sourced from different production sites (USA and Italy) on a non‐transformed small‐intestinal epithelial cell line, IEC‐6. The effects on cellular morphology, viability, migration, and H2O2‐induced damage, were assessed before and after the treatment with both VSL#3 formulations. While the USA‐sourced product (“USA‐made”) VSL#3 did not affect monolayer morphology and cellular density, the addition of bacteria from the Italy‐derived product (“Italy‐made”) VSL#3 caused clear morphological cell damage and strongly reduced cellularity. The treatment with “USA‐made” lysate led to a higher rate of wounded monolayer healing, while the addition of “Italy‐made” bacterial lysate did not influence the closure rate as compared to untreated cells. While lysates from “USA‐made” VSL#3 clearly enhanced the formation of elongated and aligned stress fibers, “Italy‐made” lysates had not similar effect. “USA‐made” lysate was able to cause a total inhibition of H2O2‐induced cytotoxic effect whereas “Italy‐made” VSL#3 lysate was unable to protect IEC‐6 cells from H2O2‐induced damage. ROS generation was also differently influenced, thus supporting the hypotesis of a protective action of “USA‐made” VSL#3 lysates, as well as the idea that “Italy‐made” formulation was unable to prevent significantly the H2O2‐induced oxidative stress. The data here reported introduce the wound‐healing assay as a tool for testing probiotics aimed at protecting gastrointestinal mucosal surfaces and to verify the consistency of their manufacturing. At the scope, we compared the in vitro effects of two multi‐strain high concentration formulations both commercialized under the same brand VSL#3 but sourced from different production sites (USA and Italy) on a non‐transformed small‐intestinal epithelial cell line, IEC‐6. Our results suggest that all the cellular parameters analyzed on a rat intestinal epithelial cell line (IEC‐6)—cell viability and death, wounded monolayer healing ability and protection against H2O2‐induced cellular injury—appear to be differently influenced by the “Italy‐made” VSL#3 when compared to the “USA‐made” VSL#3, thus reconfirming previous reports supporting that sourcing is a key element for probiotic safety and or efficacy.