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Background: Antimicrobial resistance has worsened in Latin America. There is an urgent need to understand the development of antimicrobial stewardship programs (ASPs) and the barriers to implementing effective ASPs in light of limited national action plans or policies to promote ASPs in the region., Methods: We performed a descriptive mixed-methods study of ASPs in 5 Latin American countries in March-July 2022. An electronic questionnaire with an associated scoring system (hospital ASP self-assessment) was used, and ASP development was classified based on the scores (inadequate, 0-25; basic, 26-50; intermediate, 51-75; or advanced, 76-100). Interviews among healthcare workers (HCWs) involved in antimicrobial stewardship (AS) inquired about behavioral and organizational factors that influence AS activities. Interview data were coded into themes. Results from the ASP self-assessment and interviews were integrated to create an explanatory framework., Results: Twenty hospitals completed the self-assessment, and 46 AS stakeholders from these hospitals were interviewed. ASP development was inadequate/basic in 35% of hospitals, intermediate in 50%, and advanced in 15%. For-profit hospitals had higher scores than not-for-profit hospitals. Interview data validated the self-assessment findings and provided further insight into ASP implementation challenges, which included limited formal hospital leadership support, inadequate staffing and tools to perform AS work more efficiently, limited awareness of AS principles by HCWs, and limited training opportunities., Conclusions: We identified several barriers to ASP development in Latin America, suggesting the need to create accurate business cases for ASPs to obtain the necessary funding for their effective implementation and sustainability., Competing Interests: Potential conflicts of interest. V. F. reports funding support from CDC including funding to present this work. S. E. C., reports funding support from the CDC. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)