Rationale: 3,14,19-Triacetyl andrographolide (ADA), a derivative of andrographolide, possesses promising anti-inflammatory and anti-tumor properties. However, there is currently no pharmacokinetic study of ADA. Accurate quantification of ADA in biological matrices is crucial for evaluating its pharmacokinetic profile and therapeutic potential. Here, we aimed to develop a rapid and sensitive UPLC-MS/MS method for quantifying ADA in rat plasma, facilitating preclinical pharmacokinetic studies. Methods: An ACQUITY UPLC BEH C18 column was employed for chromatographic separation of ADA and celecoxib (internal standard, IS) using a gradient mobile phase of acetonitrile/water (both containing 0.1% formic acid) at a flow rate of 0.3 mL/min. Quantification was achieved via multiple reaction monitoring (MRM) transitions of m/z 499.1→439.1 for ADA and m/z 381.9→361.9 for celecoxib. Plasma samples underwent liquid-liquid extraction prior to analysis. The method’s linearity, precision, accuracy, matrix effects, and recovery were thoroughly evaluated. Additionally, we administered different doses of ADA via two routes of administration (intragastric administration and intravenous injection) in rats and determined its plasma pharmacokinetic profile in vivo, including estimating its oral bioavailability. Results: The developed method exhibited excellent linearity (R 2=0.9921) over a concentration range of 5-2000 ng/mL for ADA in rat plasma. Both within-run and between-run precision (RSD%) were less than 6.88%, and the accuracy ranged from 94.86% to 107.61%. Matrix effects were found to be between 87.53% and 112.78%, with recoveries ranging from 98.60% to 104.39%. ADA was stable under various storage conditions in plasma. Applying this validated method to pharmacokinetic studies in rats showed that ADA exhibited linear pharmacokinetic characteristics in the dose range of 100-400 mg/kg, and the oral bioavailability was 7.193%-9.169%. Conclusion: The established UPLC-MS/MS method provides a rapid, sensitive, and reliable tool for the quantification of ADA in rat plasma, enabling comprehensive pharmacokinetic studies and further evaluation of its therapeutic potential.