Midazolam (MDZ) is effective in treating seizures in a medical emergency service. However, intravenous (i.v.) administration requires skillful trained personnel. Alternative routes such as rectal, buccal, sublingual, or intranasal administration are established choices for drug application in an out-patient service. The aim of this work was to use rabbit as a model to compare the pharmacokinetics of midazolam and its 1′-hydroxy metabolite (1′-OH-MDZ) via i.v., rectal, intranasal and buccal administration with novel formulations. A single-dose, randomized, open-label, four-period crossover pharmacokinetic study was conducted with a three-day washout period between each segment. CYP3A activities were compared by studying the enzyme kinetics of midazolam in rabbit (CYP3A6) and human (CYP3A4/5) liver microsomes to qualify rabbit as a model species to predict human metabolic activity. From this study, a comparable apparent K_m (7.86 vs. 8.66 μM) but a slightly higher V_max (2117 vs. 1361 pmol min⁻¹ mg⁻¹) was observed in rabbits, and this resulted in a 1.72-fold higher intrinsic clearance. Midazolam had comparable bioavailability among 4 routes tested (60–70%) which is higher than oral (35–44%) administration. The absorption was faster in intranasal and rectal (∼10 min) than the buccal administration (∼20 min). The in vivo study also indicated that female rabbits had around 2-fold higher activity (1′-OH-MDZ/MDZ) in CYP3A6 than the male rabbits suggested that male rabbits may be closer to human in CYP3A activity. Overall, the rectal and intranasal formulations under current development might have the potential for administering midazolam in an out-patient emergency service when i.v. administration is not an option.