Migration brings significant challenges to health and quality of life when acculturation exceeds migrants’ personal resources, causing acculturative stress. Despite its high prevalence, there are few instruments to assess it in Latin America. The aim of this study was to develop an instrument to measure acculturative stress based on a sample of 283 migrants in Chile. Twenty-five items were developed and grouped into three factors. Findings show excellent psychometric properties of reliability, validity, and cultural relevance. The instrument is appropriate for intraregional migrant populations in Latin America.