Andrographolide DMSc reference substance was produced by extraction of leaves of Andrographis paniculata (Burm.f.) Nees with chloroform. Andrographolide crude extract was purified by recrystallization with ethanol. Identification of andrographolide raw material was performed by IR spectroscopy, mass spectroscopy, 1H-NMR spectroscopy and differential scanning calorimetry to ensure the identity and purity of this substance. The assigned value of andrographolide DMSc reference substance was 98.2% calculated on the as is basis, determined by UPLC method compared with andrographolide USP reference standard. This developed method of liquid extraction and purification by recrystallization was a non-complicated method but selective separation of andrographolide from other diterpene lactones with the yield of 0.18%.