1-aminobenzotriazole (ABT) is a known P450 enzyme non-selective inactivator that serves as a tool for assessing P450-mediated metabolism. However, many findings demonstrated that ABT was ineffective with human CYP2C9. A profound pan-CYP450 inhibitor is desired avoid the risk of incomplete inhibition of P450, especially CYP2C9. Atipamezole is commonly used to recover animals from sedation-anesthesia induced by αadrenoceptor agonists. The purpose of this study is to evaluate atipamezole as a non-selective inhibitor of P450 enzymes and compare it with ABT. Inhibition toward seven major human CYP450 isoform was determined for atipamezole and ABT in human, rat, and dog liver microsomes for the direct and time-dependent inhibition potentials. IC values toward human and animal CYPs without preincubation are 0.02-7.93 μM and 20.9-1798 μM for atipamezole and ABT, respectively. The IC values of ABT after preincubation shift to 4.06-460 μM. Atipamezole has more effective inhibition to CYP2C9 mediated diclofenac hydroxylation in human and animal liver microsomes with IC values of 1.50-5.20 μM than that of ABT at 74.7-460 μM. No IC shift was observed for atipamezole to CYP isoforms. In vivo utility of atipamezole was assessed by co-dosing with diclofenac in rats. At 30 mg/kg via oral, atipamezole enhanced the AUC of diclofenac by 13.1-fold and the C by 5.6-fold. Similar enhancement also achieved for ABT (100 mg/kg) with AUC and C increased 9.5 and 4.8-fold. As a reversible pan-CYP inhibitor, atipamezole showed less species difference than ABT. It provides a better and easier to use alternative to ABT for ADME optimization and elucidating mechanistic drug metabolism or toxicity studies.