Andrographis paniculata is an important medicinal plant belongs to the family Acanthaceaepossesses numerous valuable secondary metabolites. With the growing commercial importance for secondary metabolites has led to a great demand in the pharmaceutical industryin recent years. Therefore, an efficient callus production protocol was developed as a tool for extracting valuable secondary metabolites from Andrographis paniculata. Nodal cuttings, leaves and seeds were tested for surface sterilization with two Clorox® concentrations (10%, 15 %) and three exposure time periods (5 min, 10 min, 15 min). For callus induction and multiplication, the established cultures were transferred to Murashige and Skoog (MS) medium supplemented with various combinations and concentrations of 1-Naphthaleneacetic acid (NAA) (1.0, 2.0 (mgL-1)) and 2,4-Dichlorophenoxyacetic acid (2, 4 D) (0.5, 1.0, 1.5, 2.0 (mgL-1)). Seeds were the most suitable explant for callus induction because of 100% non-contamination with all treatments. But for nodes and leaves, 0% non-contamination was observed (P ≤ 0.05). When considering minimum resources allocation, 10% Clorox® for 5 minutes exposure time period was selected as the best surface sterilization method. Highest callus formation (91.8%) was observed in MS medium supplemented with 2.0 mgL-1 2, 4 D + 1.0 mgL-1 NAA (p ≤ 0.05). Result was significantly different only from the treatments with 1.0 mgL-1 2, 4 D + 1.0 mgL-1 NAA and 1.5 mgL-1 2, 4 D + 2.0 mgL-1 NAA. Excellent callus with non-brownish colour (83%) and callus with excellent growth (67%) were observed in the same treatment. The optimized protocol could be used to produce higher amount of callus in order to extract secondary metabolites from Andrographis paniculata.