Isoflavones, known as one type of phytoestrogen with a potential anticarcinogenic activity, are found in a variety of vegetables and legumes in small quantities. However, soybean is a rich source of isoflavones ranging from 886 to 3495 mg/kg of soy flour. It has been reported that isoflavone content in soybean seeds is affected by solvents, pH, and temperature during extraction, as well as environmental and genetic factors. Natural elicitors such as salicylate applied on plants at different growth stages may affect isoflavone content in soybean seeds. The objectives of the study were: 1) to identify the best one of eight solvents for total isoflavones extraction from R96-3444, soybean with high isoflavone content; 2) to evaluate the effect of three natural elicitors with various concentrations on isoflavone content; and 3) to determine the soybean growing stage affected mostly by natural elicitors for isoflavone enhancement. The soybean plants were sprayed at R1 (blooming) and R4 (full pods) growing stages with salicylic acid, methyl salicylate, and ethyl acetate at 0, 1x10^-6M, 1x10^-3M, and 1x10^-1M concentration levels. The isoflavones extracted with various solvents including MeOH/H₂O/0.1N HCL (10:7:2), 53% MeOH/H₂O, EtOH/H₂O/0.1N HCL (10:7:2), 53% EtOH/H₂O, Acetontrile/H₂O/0.1N HCL (10:7:2), 53% Acetontrile/H₂O, H₂O (ambient temperature), and hot H₂O (60°C) were separated, identified, and quantified by an established HPLC procedure. Results showed that glycitin, genistin, M-glycitin, M-genistin, A-genistin, and total isoflavone content (4194 mg/kg) were the highest with 53% Acetontrile/H₂O solvent in comparison to other solvents. Daidzin, glyctin, genistin, M-glycitin, A-glycitin, daidzein, glycitein, and genistein content were the highest in the seeds of R96-3444 sprayed with 1x10^-3M salicylic acid at the R4 stage. Total isoflavone content of R96-3444 sprayed at the R1 stage is significantly higher (p=0.008) than that at the R4 stage, and no significant differences were observed among three natural elicitors.