Determination of major bioactive compounds in polyphyto-formulation is important for production of standardized herbal products. A fast, simple and inexpensive method for detection and quantification of andrographolide concentration in nanoemulsion preparations containing a combination of Andrographis paniculata (Burm f.) Ness. and Phyllanthus niruri L. has been developed. Detection and quantification were carried out using UV-vis spectrophotometry analysis with picric acid reagent and NaOH (8:2) in methanol solvents, read at maximum wavelength 479 nm with 22 min of incubation time. Validation was done by determine the parameters such as linearity, intra and interday precision, accuracy, limit of detection (LOD) and limit of quantification (LOQ). The results obtained showed linearity with r = 0.9945 (y = 0.0109x − 0.2066) in the range of 30-80 μg/mL. The accuracy (recovery) varied in the range of 97.15 to 104.42 %. Percentage of relative standard deviation (% RSD) for precision and intermediate precision value were 3.23 and 3.02 % with LOD value 211 μg/mL and the LOQ 705 μg/mL. As a conclusion, this method is suitable to detect andrographolide content in herbal nano-preparation. Keywords: Andrographis paniculata, Phyllanthus niruri, Spectrophotometry, Andrographolide, Nanoemulsion.