AbstractApproaches and guidelines for performing subgroup analysis to assess heterogeneity of treatment effect in clinical trials have been the topic of numerous papers in the statistical and clinical literature, but have been discussed predominantly in the context of conventional superiority trials. Concerns about treatment heterogeneity are the same if not greater in non‐inferiority (NI) trials, especially since overall similarity between two treatment arms in a successful NI trial could be due to the existence of qualitative interactions that are more likely when comparing two active therapies. Even in unsuccessful NI trials, subgroup analyses can yield important insights about the potential reasons for failure to demonstrate non‐inferiority of the experimental therapy. Recent NI trials have performed a priori subgroup analyses using standard statistical tests for interaction, but there is increasing interest in more flexible machine learning approaches for post‐hoc subgroup discovery. The performance and practical application of such methods in NI trials have not been systematically explored, however. We considered the Virtual Twin method for the NI setting, an algorithm for subgroup identification that combines random forest with classification and regression trees, and conducted extensive simulation studies to examine its performance under different NI trial conditions and to devise decision rules for selecting the final subgroups. We illustrate the utility of the method with data from a NI trial that was conducted to compare two acupuncture treatments for chronic musculoskeletal pain.