Talinum paniculatum (Jacq.) Gaertn has long been used as a medicinal plant in Indonesia. It has similar root morphology to Panax ginseng. Leaves of T.paniculatum is known as a source of flavonoids. It has antioxidant and antibacterial activities. In vitro culture is one of the tools in Biotechnology that can elevate secondary metabolites in cells by adding an elicitor. Methyl Jasmonate is a biotic elicitor that is naturally produced by plants as a defensive response against pathogens. Factors that affect the production of biomass and secondary metabolites are the concentration of elicitor and exposure (elicitation) duration. Therefore, this study aimed to evaluate the effect of methyl jasmonate concentration (0,50,100, and 150) µM and elicitation duration (0, 48, 96, 144) hours on callus biomass and flavonoid production of T.paniculatum. The results showed that the increase of methyl jasmonate concentration and duration of elicitation caused a decrease in callus biomass compared to the control. The highest callus (dry weight) biomass (0.062 g/g DW) was recorded in callus cultures elicited in 150 µM methyl jasmonate for 96 hours. Considering the flavonoid content, 150 µM methyl jasmonate with the duration of elicitation of 96 hours also produced the highest flavonoid content measured by TLC stained area (0.121 cm2). It is concluded that the higher concentration of methyl jasmonate and the longer duration of elicitation influenced the inhibition of callus growth but increase the production of flavonoids.