Anthropogenic global climate change is contributing to the mass extinction of wildlife across the globe. The protection of biodiversity in freshwater environments ensures economically beneficial ecosystem services are maintained. Freshwater ecosystems are hotspots for biodiversity and have higher levels of imperiled organisms compared to terrestrial and marine systems. The objective of this study was to develop an aquatic environmental DNA (eDNA) protocol that increases detection sensitivity to target rare and endangered aquatic organisms in Kansas. The Cylindrical Papershell mussel (Anodontoides ferussacianus) was listed as endangered in Kansas in 2019 and was used for protocol development due to its contracting distribution and low population density. While this species was used in the development of the protocol, the objective of this protocol is not restricted to this species. Following this protocol, Cylindrical Papershell DNA was detected at a sensitivity of 1.7 parts per billion in 1 L of water. Increasing detection sensitivity will be necessary for successful conservation strategies as we proceed through the Anthropocene due to compounding costs associated with monitoring and managing an increasing number of declining populations. This protocol, in conjunction with traditional sampling efforts, will aid in future monitoring and recovery plans of native aquatic species.