Objective · To perform the pharmacokinetic (PK) studies of DDDA3, a dual inhibitor of both acetylcholinesterase and butyrylcholinesterase, into provide experimental evidence for the evaluation of in vivo activity against Alzheimers disease (AD) and the formulation optimization. Methods · A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the quantification of DDDA3 was developed and validated. This method was applied to pharmacokinetic studies of DDDA3 in rat plasma. Protein precipitation approach was used to prepare plasma samples. To lower the detection limit, the concentration step was utilized. The samples were analyzed on a Zorbax SB-Aq column (2.1 mm×100 mm, 3.5 μm) with the mobile phase composed of methanol and water (75:25volume, containing 0.1% formic acid and 10 mmol/L ammonium formate) and determinedpositive electrospray ionization in multiple reaction monitoring mode. Results · The lower limit of quantification was 0.5 ng/mL and the linear range was 0.5-500 ng/mL. The absolute oral bioavailability of DDDA3 in SD rats was 78.4%. Conclusion · DDDA3 has high potentials as anti-AD agents, with good oral bioavailability, fast absorption rate, and long-lasting blood concentration; however, the formulation should be optimized to improve the solubility.