Improving long-term kidney transplant outcomes requires novel treatment strategies, including delayed calcineurin inhibitor (CNI) substitution, tested using informative trial designs. An alternative approach to the usual superiority-based trial is a non-inferiority trial design that tests whether an investigational agent is not unacceptably worse than standard of care. An informative non-inferiority design, with biopsy-proven acute rejection (BPAR) as the endpoint, requires determination of a pre-specified, evidence-based non-inferiority margin for BPAR. No such information is available for delayed CNI substitution in kidney transplantation. Herein we analyzed data from recent kidney transplant trials of CNI withdrawal and “real world” CNI- based standard of care, containing subjects with well-documented evidence of immune quiescence at 6-months post-transplant-ideal candidates for delayed CNI substitution. Our analysis indicates an evidence-based non-inferiority margin of 13.8% for the United States Food and Drug Administration’s composite definition of BPAR between 6- and 24-months post- transplant. Sample size estimation determined that ~225 randomized subjects would be required to evaluate non-inferiority for this primary clinical efficacy endpoint, and superiority for a renal function safety endpoint. Our findings provide the basis for future delayed CNI substitution non-inferiority trials, thereby increasing the likelihood they will provide clinically implementable results and achieve regulatory approval.This article is protected by copyright. All rights reserved.