This report describes the rationale for and application of a protocol for estimation of ecologically-relevant streamflow metrics that quantify streamflow regime for ungaged sites subject to a range of human impact. The analysis presented here is focused on sites sampled by the U.S. Environmental Protection Agency as part of their Environmental Monitoring and Assessment Program in the upper Missouri Basin and Oregon. Streamflow data are provided by the U.S. Geological Survey. Specific guidance is provided for selection of gage sites, development of probabilistic frequency distributions for annual peak- and 7-day low-flow events, and regionalization of the frequency curves based on multivariate analysis of watershed characteristics. Evaluation of the uncertainty associated with the various components of this protocol indicates that the results are reliable for the intended purpose of hydrologic classification to support ecological analysis. They should not be considered suitable for more standard water-resource evaluations that require greater precision, especially those focused on management and forecasting of extreme flow conditions.