Andrographolide (AGP) is the main bioactive component of Andrographis paniculata. It is an important medicinal plant used in the traditional medicine for the treatment of many infectious diseases. AGP has an interesting pharmacophore but its poor water solubility causes lower bioavailability, seriously limited its pharmacological function after oral administration. The objectives of this study were to prepare the self microemulsifying drug delivery system (SMEDDS) formulation to improve the oral bioavailability of AGP and to develop an analytical method to determine its pharmacokinetic parameters. An analytical method using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was established to quantify AGP in rat plasma. Protein precipitation technique was used to extract AGP from the plasma and then separated on an HSS C18 column with a mobile phase consisting of methanol–water (70:30). Pharmacokinetic investigations were performed in three groups of rats each group consists of 4 rats following oral administrations of unformulated and SMEDDS formulation of AGP (10 mg/kg) and intravenous administration of AGP (2 mg/kg). Cmax of SMEDDS formulation was 5.8-fold greater than Cmax of AGP unformulated. Tmax was decreased about 21 min. AUC0-24 of SMEDDS formulation increased by 5.1-fold compared to the unformulated AGP. SMEDDS formulation improved the absolute oral bioavailability of AGP from 3.3% to 16.7% which was a 5.1-fold increase.