Little research has taken place on the effect of euthanasia methods on biophysical and biochemical changes at the time of euthanasia in fish. These changes are used in multiple species to determine stress levels before death. Koi (Cyprinus carpio) are an important fish species often used in laboratory research, kept in backyard ponds, and managed in zoological and aquarium collections. The current study evaluated euthanasia of koi by immersion in 0.5 g/L tricaine methanesulfonate (MS-222) (n = 10), 0.5 g/L clove oil (n = 8), 1 g/L clove oil (n = 10), and CO₂ (n = 7) on time to cessation of opercular movement, plasma lactate levels, and plasma cortisol levels. CO₂ had the longest mean time to cessation of opercular movement, and MS-222 had the shortest (mean CO₂: 24.9 min, range 13.18–31.35 min; MS-222: 2.68 min, range 1.33–4.5 min). The difference was not significant between any of the groups for plasma cortisol or lactate levels. MS-222 demonstrated the highest cortisol levels, and CO₂ had the lowest (mean CO₂: 108.7 ng/ml, range 33.9–195.8 ng/ml; MS-222: 650.6 ng/ml, range 77.3–2374.9 ng/ml). Average lactate levels were highest for 1 g/L clove oil and lowest for 0.5 g/L clove oil (mean 0.5 g/L clove oil: 5.1 mmol/L, range 1.8–8.1 mmol/L; 1 g/L clove oil: 7.4 mmol/L, range 5.6–10.5 mmol/L).