Identification of reference streams and human disturbance gradients are crucial steps in assessing the effects of human disturbances on stream health. We describe a process for identifying reference stream reaches and assessing disturbance gradients using readily available, geo-referenced stream and human disturbance databases. We demonstrate the utility of this process by applying it to wadeable streams in Michigan, USA, and use it to identify which human disturbances have the greatest impact on streams. Approximately 38% of cold-water and 16% of warm-water streams in Michigan were identified as being in least-disturbed condition. Conversely, approximately 3% of cold-water and 4% of warm-water streams were moderately to severely disturbed by landscape human disturbances. Anthropogenic disturbances that had the greatest impact on moderately to severely disturbed streams were nutrient loading and percent urban land use within network watersheds. Our process for assessing stream health represents a significant advantage over other routinely used methods. It uses inter-confluence stream reaches as an assessment unit, permits the evaluation of stream health across large regions, and yields an overall disturbance index that is a weighted sum of multiple disturbance factors. The robustness of our approach is linked to the scale of disturbances that affect a stream; it will be less robust for identifying less degraded or reference streams with localized human disturbances. With improved availability of high-resolution disturbance datasets, this approach will provide a more complete picture of reference stream reaches and factors contributing to degradation of stream health.