In this systematic review we sought to identify how the public health literature focusing on Hispanic populations in the United States defined and measured the concept of acculturation. A review of 134 studies found considerable variation in the definition and measurement of this construct. The ten acculturation scales used provided little theoretical orientation. It was unclear the extent to which acculturative changes in attitudes, beliefs and behaviors were captured by current measurement tools, as these primarily measure linguistic elements. We suggest future research should refine existing tools, determine their validity and usefulness across ethnic and subethnic groups, and identify which aspects of acculturation these scales and indices reliably measure. Recommendations for use of acculturation instruments in public health practice with Hispanic populations are included.