Andrographis paniculata (Acanthaceace) commonly known as “kalmegh” and also called as “king of bitters” is used from ancient time. The main active constituents present in Andrographis paniculata is Andrographolide. The present study deals with comparative study of assay by HPTLC method and antibacterial activity for Andrographis paniculata extract at accelerated storage condition for a period of 6 months. The antibacterial activity of methanolic extract of dried stems of Andrographis paniculata was determined by broth dilution method against gram-positive bacterial strain (Staphylococcus aureus) and gram-negative bacterial strain (Escherichia coli). For HPTLC method, the active marker, andrographolide and extract was spotted on the plates precoated with silica gel 60 F254 and developed using chloroform: methanol(9:1v/v) as mobile phase. Densitometric analysis was carried out at 226 nm. The method showed high sensitivity with good linearity over the concentration range of 200-1000ng/spot. The peak for andrographolide was observed at Rf of 0.34 ±0.02. The aim of our study was to observe the effect of accelerated storage on assay of pure marker and marker in the extract. The analysis was carried out at 1,2,3,6 months study as per ICH guidelines for stability testing of drug at storage condition of 40ºC ± 2ºC/75% RH ± 5% RH.