This paper presents, for the first time, electrochemical behavior of diterpene lactone andrographolide and its voltammetric determination using differential pulse voltammetry (DPV) at boron-doped diamond electrode, in human urine and oil extract from Andrographis paniculata seeds. Andrographolide, due to its chemical structure, provides well defined peak at + 1.55 V vs. Ag/AgCl electrode (3 M) in Britton–Robinson buffer solution pH 12. The proposed method, under optimum experimental parameters displays excellent selectivity towards andrographolide in the presence of some interference, as ascorbic acid, uric acid and dopamine, with detection limit of 0.75 μM. Calibration curves were found to be linear in the range from 2 to 40 μM, with two linear ranges, first from 2 to 10 μM and second from 10 to 40 μM with corresponding equations I (μA) = (4.00 ± 0.12) c (μM) + (13.42 ± 0.66) (R2 = 0.9975) and I (μA) = (0.87 ± 0.05) c (μM) + (46.33 ± 1.53) (R2 = 0.9767), respectively. Relative standard deviation of 7 measurements was 4% which verified excellent repeatability of proposed method and this method was successfully applied for the determination of andrographolide in human urine sample and extract from the A. paniculata seeds. The Prussian blue method was used for validation of the proposed elactroanalytical method