The Ultra Performance Liquid Chromatography method was developed and validated using a diode array detector and a mass spectrometer to determine the content of the andrographolide. The hromatographic conditions were: ACQUITY® UPLC BEH C18, 1.7μ column, size 2.1 x 100 mm, mixture of 0.050% formic acid in water and 0.025% formic acid in acetonitrile as the mobile phase of the gradient system, flow rate 0.5 ml/min and UV detector 223 nm. The chromatogram showed suitability of chromatographic system, which conformed to the United States Pharmacopoeia 39th revision (USP39) requirements. The selectivity and specificity of the method were assessed by the separation of andrographolide from the other diterpene lactones. The developed method showed good accuracy, precision, linearity and range including robustnesss, according to the USP and ICH guidelines. The validated method could be used as the reference method for the assay of the andrographolide substance in andrographolide bulk material and powdered andrographis.