Euthanasia of laboratory animals is among the fundamentals of animal experimental ethics. Apart from the widely accepted euthanasia method involving anesthesia, non-anesthesia method are also commonly used. On the basis of the “ 3R” principle of animal experimental ethics and the “ five freedoms” of experimental animal welfare, we systematically discuss the ethical legitimacy of two non-anesthetic euthanasia method, decapitation and cervical dislocation. Additionally, potential ethical problems and possible improvements of air embolism, liquid nitrogen freezing, bloodletting, and apnea as euthanasia method are discussed. We also propose feasible approaches of euthanasia involving integrative method. Our analysis may assist researchers in performing ethical non-anesthesia euthanasia.