This chapter explores the optimization of biotic elicitation for enhancing capsaicin production in Capsicum annuum suspension cultures using Aspergillus niger fungal cultures. The protocol involves preparing MS and B5 media, establishing callus cultures from aseptic seedling explants, and maintaining liquid cultures. Fungal elicitors are prepared by growing Aspergillus niger on a nutrient medium, extracting the fungal mat and cell filtrates, and applying these to the cultures. Different concentrations of fungal extracts are tested during the log phase of growth, and the effects on Packed Cell Volume (PCV), fresh weight, dry weight, and capsaicin content are measured at regular intervals. The results indicate that optimal concentrations and timing of elicitor application significantly enhance capsaicin production, providing a valuable method for improving secondary metabolite yields in plant cultures. This optimized protocol can potentially be applied to other plant species and secondary metabolites.