Background: PI-301 is an oral asthma drug candidate that binds GABAA receptors on airway smooth muscle and immune cells. Objective: The objective of this study is to identify and quantify PI-301 metabolites in vitro and in vivo and determine the pharmacokinetics of oral, IP, and IV administrated PI-301. Methods: In vitro conversion of PI-301 was performed using liver and kidney microsomes/S9 fractions followed by quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A LC-MS/MS method was developed using synthesized standards to quantify PI-301 and its metabolites in urine and feces. Blood, lung, and brain were harvested from animals that received PI-301 by oral, IP, and IV administration, followed by LC-MS/MS quantification. Imaging mass spectrometry was used to demonstrate the presence of PI-301 in the lung after oral administration. Results: PI-301 is stable in the presence of liver and kidney microsomes and S9 fractions for at least two hours. PI-301 undergoes conversion to the corresponding glucuronide and glucoside in the presence of conjugating cofactors. For IP and IV administration, unconjugated PI-301 together with significant amounts of PI-301 glucoside and PI-301 taurine were found in urine and feces. Less conjugation was observed following oral administration, with PI-301 glucuronide being the main metabolite. Pharmacokinetic quantification of PI-301 in blood, lung, and brain showed very low levels of PI-301 in the brain after oral, IV, or IP administration. The drug half-life in these tissues ranged between 4-6 hours for IP and oral and 1-2 hours for IV administration. Imaging mass spectrometry demonstrated that orally administered PI-301 distributes uniformly in the lung parenchyma. Conclusion: PI-301 undergoes no phase I and moderate phase II metabolism.