Andrographolide (AG) have recently attracted considerable interest because of their diverse physiological functions and therapeutic potential. As pointed out by the WHO there is very limited knowledge about the pharmacokinetics, pharmacodynamics, and metabolomics of Indian medicinal plants (IMP). The objective of the present work was to develop a simple, accurate, sensitive RP-HPLC method with appropriate validation for determination and quantification of AG and its formulation in rat plasma. Separation and detection of andrographolide from herbal formulation was achieved on reversed phase HPLC column using acetonitrile: water (40: 60 v/v). The method was validated as per the norms of the ICH guidelines and applied to study the pharmacokinetics of andrographolide in commercial formulation in terms of bioavailability. The HPLC method validation has been shown a linear calibration curve over a plasma concentrations range of 2 to 12 µg/mL with a correlation coefficient of 0.9979, the limit of detection and the limit of quantification were determined to be 0.025 and 0.07µg/ml respectively. The absorption and elimination profile of AG from formulation was developed by oral administration in rat. The method was found to be sensitive, accurate and reproducible. Therefore, it can be recommended for marker-based standardization and quality assurance and pharmacokinetic profile of A.paniculata and its traditional preparations.