Carbon dioxide (CO2) administration is the most commonly used method of euthanasia of mice in research, yet questions remain regarding whether CO2 euthanasia is associated with pain and stress. This study aims to characterize the level of pain and stress induced in mice during CO2 euthanasia, and to determine if premedication with acepromazine or midazolam, or anesthetic induction with isoflurane, alters these levels during CO2 euthanasia. Mice were assigned to one of six euthanasia groups: (control) CO2 only at a flow rate that displaces 20% of the cage volume per minute (V/min); premedication with acepromazine (5 mg/kg), midazolam (5 mg/kg), or saline followed by 20% V/min CO2; induction with 5% isoflurane followed by > 100% V/min CO2; or 100% V/min CO2 only. Behavioral measures of stress included ultrasonic sound recordings and analysis of video recordings, by an observer blinded to group identity, to assess increased respiratory effort, increased activity, and pain. Physiological parameters of stress were assessed by measuring plasma adrenocorticotropic hormone (ACTH) and corticosterone levels immediately post-euthanasia. Finally, we assessed the acute neuromolecular marker of pain and stress, c-fos, by quantitative PCR. The use of premedication with acepromazine or midazolam did not significantly alter behavioral indicators of stress but did significantly induce a higher level of c-fos expression in the brain compared to 20% V/min CO2 alone. Furthermore, the use of isoflurane induction prior to CO2 euthanasia significantly increased stress in the mice based on both behavioral and neuromolecular indicators. These data strongly indicate that in comparison to the other modalities analyzed in this study, 20% V/min CO2 is a humane, rapid euthanasia method that is not associated with significant pain or stress in mice.