Background: Rotator cuff tears (RCTs), resulting from degeneration or trauma of the shoulder tendons, are one of the main causes of shoulder pain. In particular, massive RCTs represent 40% of all injuries, require surgical treatment, and are characterized by poor clinical outcomes and a high rate of failure. In recent years, the use of biological decellularized patches for augmentation procedures has received great interest owing to their excellent self-integration properties, improving healing and, thus, presenting an innovative therapeutic option. However, the findings from clinical studies have emerged with conflicting viewpoints regarding the benefits of this procedure, as an excessive tension load might compromise the integrity of the tendon-to-bone connection when the patch exhibits low elasticity or insufficient strength. This could prevent the healing process, leading to unpredictable results in clinical practice.Methods: This systematic review was conducted following Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines across three databases (PubMed, Scopus, and Web of Knowledge) to underline the results obtained in preclinical studies involving animal models of RCT surgeries that utilized the biological decellularized matrix augmentation technique in the last 5 years.Results: Thirteen articles were included after the screening, and the SYRCLE tools were applied to assess the risk of bias in in vivo studies. Open-surgery techniques were conducted to create tendon defects or detachment in different animal models: rat (31%), rabbit (46%), dog (15%), and sheep (8%). Patches decellularized with non-standardized protocols were used in 77% of studies, while commercially available matrices were used in 15%. Of the studies, 31% used allogenic patches, 61% used xenogenic patches, and 8% utilized both xenogenic and autologous patches.Conclusion: Overall, this review provides a comprehensive overview of the use of acellular patches and their effective therapeutic potential in rotator cuff (RC) repair at the preclinical level with the aim of expanding the strategies and matrices available for surgeons.Systematic review registration:https://www.crd.york.ac.uk/prospero/, identifier CRD42023468716.