Biological monitoring in the Rouge Education Project focuses on collecting and identifying benthic macroinvertebrates. These are bottom-dwelling, invertebrate organisms visible to the naked eye, such as aquatic insect larvae, crayfish, snails, leeches and aquatic worms. The types and numbers of macroinvertebrates living in the river indicate the water quality of an area. Some macroinvertebrates are very sensitive to pollution and are only found in pristine areas, while others have a high tolerance for pollution and can live in lower quality areas.

In the Rouge Education Project, students or their teachers collect macroinvertebrates in the river with a net (while wearing waders), and then identify the organisms onshore using reference materials provided by the project. They document their findings on a form that allows them to calculate a stream quality score. Teachers may also choose to undergo special training in benthic macroinvertebrate collection and identification that enables the Michigan Department of Environmental Quality to utilize their data.