The objective of this study was to compare the sedative effects of intramuscular alfaxalone combined with either ketamine or midazolam in chickens (Gallus gallus domesticus). A prospective, randomized blinded crossover study design with a 7-day washout period was used. Nine adult layer hens received alfaxalone 15 mg/kg with ketamine 5 mg/kg IM (treatment AK) or alfaxalone 15 mg/kg with midazolam 1 mg/kg IM (treatment AM). Time to lateral recumbency, time to loss of righting reflex, induction quality, duration of loss of righting reflex, time to sternal recumbency or vigorous response to stimulation, and time to standing were recorded. Muscle tone, response to noxious stimulation, heart rate, respiratory rate, and oxygen saturation were monitored once the righting reflex was absent. Induction and recovery times were not different between treatments. Lateral recumbency was induced in 8 of 9 birds receiving AK compared to 6 of 9 birds receiving AM. Righting reflex was absent in 7 of 9 and 5 of 9 chickens administered AK and AM, respectively. Median time to loss of righting reflex for AK and AM were 5.5 (4.3–9.3) minutes and 9.1 (4.8–15.0) minutes, respectively (P = .88). Median duration of loss of righting reflex was 21.6 (16.0–36.9) minutes for AK and 21.1 (11.9–26.4) minutes for AM (P = .38). Alfaxalone-ketamine resulted in moderate excitation during induction. Further investigations are warranted to investigate the effects of alfaxalone and midazolam or ketamine at different doses.