Plethysmography is used in nonhuman primates (NHPs) to measure minute volume before aerosol exposure to an agent to calculate total time necessary in the exposure chamber. The consistency of respiratory parameters during the entire exposure time is paramount to ensuring dosing accuracy. Our study sought to validate an alfaxalone-midazolam (AM) anesthetic combination for use in aerosol studies. We hypothesized that AM would provide an adequate duration of anesthesia, achieve and maintain steady state minute volume (SSMV) for 20 min, and have anesthetic quality and side effects comparable to or better than either tiletamine-zolazepam (TZ) and ketamine-acepromazine (KA), the most common anesthetics used for this purpose currently. Two groups of NHPs, one consisting of 15 cynomolgus macaques and one of 15 rhesus macaques, received 3 intramuscular anesthetic combinations (AM, TZ, and KA), no less than one week apart. Anesthetized NHPs were placed in a plethysmograph chamber and their minute volumes were measured every 10 s to determine whether they had achieved SSMV and maintained it for at least 20 consecutive min. Achieving and reliably maintaining an SSMV for at least 20 min facilitates precise aerosol dosing of a challenge agent. Quality of anesthesia, based on the NHP's ability to achieve and maintain SSMV, was higher with AM compared with TZ and KA in both species, and AM had a longer duration of SSMV as compared with TZ and KA in cynomolgus macaques. Average SSMV was larger with AM compared with TZ in cynomolgus macaques, but larger with KA compared with AM in rhesus macaques. Duration of anesthesia was sufficient with all combinations but was longer for TZ than both AM and KA in both species. These results suggest that the AM anesthetic combination would produce the most accurate dosing for an aerosol challenge.