The Endangered Species Act requires that the Sea Lamprey Control Program (Program) ensures that its actions do not jeopardize federally listed species. Often, lampricide toxicity has not been determined and a listed species cannot be sacrificed to test. This paper reviews approaches taken to investigate the risk of lampricide exposure to the Hungerford’s crawling water beetle (Hungerford’s; Brychius hungerfordi), piping plover (Charadrius melodus), and snuffbox (Epioblasma triquetra), and describes adaptations implemented by the Program to protect these species based on the findings detailed here and in other reports and publications. For the Hungerford’s, tests were conducted to determine the toxicity of TFM (3-trifluoromethyl-4-nitrophenol) to a surrogate (Haliplus sp.). Haliplus were resistant to 3.9 times the TFM concentration required to kill larval sea lampreys. However, because avoidance behaviors were observed, it was determined that Hungerford’s would be adversely affected. Surveys demonstrated that TFM exposure during the treatment did not negatively affect the population. For the piping plover, a No Observable Adverse Effects Level, estimated from data results on the toxicity of TFM and TFM-1% niclosamide [5-Chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxybenzamide] to other bird species, was compared to an estimated daily dietary uptake using residue concentrations in burrowing mayflies (Hexagenia limbata). It was determined that neither lampricide would pose a risk to piping plover adults or chicks. Toxicity tests conducted on snuffbox (glochidia, juveniles), and ellipse (surrogate, Venustaconcha ellipsiformis; glochidia, juveniles, adults) demonstrated that TFM would not adversely affect the mussels. Surveys and field observations supported the determinations for the three species.