Plukenetia volubilis or commonly known as sacha inchi is reported to produce wide range of health-promoting bioactive metabolites. These metabolites functions as supplements in eradicating various types of diseases. Sacha inchi has large edible seeds that are rich in phenolic content, minerals and essential fatty acid, such as omega 3 (ω-3), omega 6 (ω-6), omega 7 (ω-7), and omega 9 (ω-9). In vitro cultures could serve as alternative in producing many essential sacha inchi bioactive compounds. As an initial step towards initiating in vitro culture, the effect of 2,4-D and TDZ on inducing callus cultures were investigated. Two different explants, sacha inchi leaf and male flower were used in this study. Surface sterilization of sacha inchi was first optimized to overcome culture contamination. The most effective surface sterilization is by using 70% ethanol (30 seconds) and 0.5% sodium hypochlorite (8 minutes), which resulted in 82.5% and 95% survival rate for leaf and flower explants respectively. Next, for calli induction the explants were cultured on MS medium supplemented with different concentrations of 2,4-D and TDZ, either alone or in combination and grown at 24 hours dark photoperiods. The morphology and size of callus were observed. The results obtained from the experiment varied depending on the treatments, producing either friable or compact calli of creamy white, pure white or brownish colour. For both, leaf and male flower explants, MS medium supplemented with 3% (w/v) sucrose in combination with 1.0 mg/L 2,4-D and 0.005 mg/L TDZ recorded the best response in term of callus size, forming friable creamy white callus.