Plants are the main source for obtaining secondary metabolites that are used in the pharmaceutical, cosmetic and food industries. In nature, the performance of bioactive compounds is affected by biotic and abiotic factors, an alternative to overcome this adversity is in vitro plant cultures and particularly plant cell culture that has multiple advantages, highlighting the possibility of controlling variables to increase the content. of compounds of interest. The objective of this research was to determine and optimize the effect of some elicitors on the production of secondary flavonoid metabolites in suspension cell cultures of Thevetia peruviana on a shake flask scale. The experimental part was performed from cell cultures in suspension cells of T. peruviana maintained by the Bioconversion Laboratory of the Universidad Nacional de Colombia, sede Medellin. Firstly, methyl jasmonate (MeJa) 3 µM and salicylic acid (SA) 300 µM were added to two los of cell cultures, that circumstantially they differed in their time suspension state lot 1 (9 months) and lot 2 (3 months). Secondly, certain were evaluated, in order to be optimized; the operational parameters cosidered were: day of addition, concentration and hours of harvest. Finally, the combined effect of MeJa (0.3 µM) and SA (100 µM) was determined in different proportions, the day of elicitation and the hours of harvest were constant. Flavonoid content was quantified by UV-vis spectrophotometry usinfg the AlCl3 complexation method and was evaluated at extracellular and intracellular level. In the first part, at intracellular level, SA generated 14.6 % and 9.56 % more flavonoid content than MeJa in lots 1 and 2, respectively. In the second part, 4.14 mg EQ/g DW were generated with MeJa 0.3 µM, elicitation day 5 and harvest at 90 h; 3.75 mg EQ/g DW were generated with 100 µM SA, elicitation day 0 and harvests at 96 h. In the third part, 4.62 mg EQ / g DW were generated with the combination of MeJa (0.3 µM) – SA (100 µM) in a ratio of 20-80, elicitation on day 0 and harvest at 96 h. Eliciting cell cultures of Thevetia peruviana with MeJa and SA under optimal conditions of concentration, day of addition and hours of harvest increased the content of flavonoid compounds. The results obtained could serve as a basis for the development of investigations at the bioreactor scale.