Background: 1-Aminobenzotriazole (ABT) is a well-known non-specific and mechanism-based inactivator of major cytochrome P450 (CYP) enzymes. It is used in vivo to differentiate poor absorption and extensive CYP metabolism of compounds as a reason for poor oral bioavailability, to differentiate between parent and metabolite-mediated toxicity etc. Even though ABT is administered either 2 h or 16 h before the administration of CYP substrates, there are not many reports describing the effect of these two dosing regimens on the oral exposure of CYP substrates. In addition, ABT is also reported to reduce gastric emptying. Purpose: Purpose of the study was to evaluate the effect of 2 h and 16 h pre-treatment regimens of ABT on the exposures of triazolam in rat. Another objective was to evaluate the effect of ABT on gastric emptying using acetaminophen as the absorption marker. Methods: Effect of ABT on triazolam metabolism: Rats were divided into 3 groups (n = 4/group). Animals in Groups I, II and III were administered orally with vehicle, 100 mg/kg, ABT (2 h prior) and 100 mg/kg ABT (16 h prior) to triazolam, respectively. Triazolam (2 mg/kg) was administered orally to all groups following the pretreatment. Effect of ABT on acetaminophen absorption in rat: Animals in Groups I, II and III were administered orally with vehicle, 100 mg/kg, ABT (2 h prior) and 100 mg/kg ABT (16 h prior) to acetaminophen treatment, respectively. Acetaminophen (10 mg/kg) was administered orally to all groups following the pretreatment. Blood samples were collected up to 30 h, plasma was separated and analyzed by liquid chromatography-tandem mass spectrometry. Results: Plasma area under the curve (AUC) of triazolam was increased by ~90-fold and 72-fold for the rats pre-treated with ABT at 2 h and 16 h, respectively as compared to vehicle treated animals. Similarly, the maximum plasma concentration was also increased by 12-fold and 11-fold with 2 h and 16 h pretreatment, respectively. Time to reach maximum concentration was 0.3 h, 4.8 h and 3.7 h in control, 2 h and 16 h pretreatment animals, respectively. In the case of acetaminophen, the calculated mean absorption time in control, 2 h and 16 h ABT pretreatment groups were 0.3, 6.2 and 3.3 h, respectively, suggesting delayed absorption of acetaminophen. Conclusion: The plasma exposure of triazolam was increased to a similar extent with both 2 h and 16 h ABT pretreatment regimens. ABT delayed the absorption of acetaminophen. However, the effect was less when ABT was dosed 16 h prior compared to 2 h pretreatment.