Quantifying the effects of streamflow alteration on assemblage response is central to understanding the role humans play in shaping aquatic environments. These changes represent a level of complexity that impedes developing quantitative links between flow and ecological response because stream hydrology is strongly intertwined with natural and anthropogenic factors. Better management outcomes require disentangling these linkages. Benthic macroinvertebrate data were combined with GIS-derived natural and anthropogenic basin characteristics to identify factors associated with changes in flow processes and assemblage characteristics. Models linking streamflow metrics and macroinvertebrate response at basin and subregion scales were developed using boosted regression tree (BRT) analysis. Basin-scale BRT analyses revealed that links between macroinvertebrate response and flow metrics were often obscured, whereas more homogeneous subregions were better able to discern relations with flow. Urban land cover was the primary factor accounting for changes in flow characteristics. Elevation, land cover, and high flow frequency were the principal variables driving changes in assemblage structure within subregions. Assemblage metrics and traits were equally useful for building response models and were affected similarly by streamflow alteration. Results indicate that response models should be developed based on upland and coastal subregions. However, when defining subregions, care should be taken to maintain data sufficiency. Developing practical flow-protection standards that support a balance between human water requirements and ecological integrity requires models that reduce uncertainty and identify management-relevant drivers. However, effective management often differs by location and models developed at the subregion level may be more applicable to management and stakeholder interests.