Background & Aim: Rubiatinctorum is one of the most well-known medicinal plants whose alizarin and other anthraquinones which are outstanding color agents with some trace of pharmaceutical properties are isolated from it. The objective of this study was to optimize alizarin production in intact plant of R.tinctorum by induction with biotic elicitors. Experimental: To increase the synthesis of alizarin, bacterial (Staphylococcus aureus and Bacillus cereus) and fungal (Fusarium oxysporum and Aspergillus fumigatus) elicitors were injected to the intact plants directly by fine needles, named elicitation-infiltration method. Then samples were analyzed through standard addition method by UV-visible spectroscopy. Results: Staphyloccus aureus (1McFarland) was the most effective one on biomass accumulation. Furthermore, among fungal elicitors Aspergillusfumigatus (0.4 mg total sugar/mL) revealed the most significant help for biomass increase. Applying bacterial elicitors imposed a dramatic increase in alizarin yield in all concentrations. The most marked increase (5 fold) was for 0.5 McFarland of Bacillus cereus. In addition, Fusarium oxysporum indicated outstanding results for alizarin production’s enhancement. Recommended applications/industries: To the author’s knowledge, the application of elicitation-infiltration method for increasing the alizarin production is studied for the first time and according to the reported results, it can be a useful method for more investigations about improvement of secondary metabolites production in other plants. Manuscript profile