In order for habitat restoration in regulated rivers to be effective at large scales, broadly applicable frameworks are needed that provide measurable objectives and contexts for management. The Ecological Limits of Hydrologic Alteration (ELOHA) framework was created as a template to assess hydrologic alterations, develop relationships between altered streamflow and ecology, and establish environmental flow standards. We tested the utility of ELOHA in informing flow restoration applications for fish and riparian communities in regulated rivers in the Upper Tennessee River Basin (UTRB). We followed the steps of ELOHA to generate univariate relationships between altered flows and ecology within the UTRB. By comparison, we constructed multivariate models to determine improvements in predictive capacity with the addition of non-flow variables. We then determined whether those relationships could predict fish and riparian responses to flow restoration in the Cheoah River, a regulated system within the UTRB. Although ELOHA provided a robust template to construct hydrologic information and predict hydrology for ungaged locations, our results do not suggest that univariate relationships between flow and ecology (step 4, ELOHA process) can produce results sufficient to guide flow restoration in regulated rivers. After constructing multivariate models, we successfully developed predictive relationships between flow alterations and fish/riparian responses. In accordance with model predictions, riparian encroachment displayed consistent decreases with increases in flow magnitude in the Cheoah River; however, fish richness did not increase as predicted 4 years after restoration. Our results suggest that altered temperature and substrate and the current disturbance regime may have reduced opportunities for fish species colonization. Our case study highlights the need for interdisciplinary science in defining environmental flows for regulated rivers and the need for adaptive management approaches once flows are restored.