Amsonia orientalis Decne. (syn. Rhazya orientalis (Decne.) A. DC.) is only cultivated as an ornamental plant but also has a medicinal merit. The natural populations of the species were taken under conservation as per the Bern Convention by European Council. As one of the most common plant growth regulators and synthetic agricultural fertilizers, the effects of 6-benzylaminopurine (BAP) at different concentrations on primary metabolites of in vitro propagated A. orientalis have been investigated by using Fourier transform infrared (FT-IR) spectroscopy. Among all applied concentrations, 2.0 mg l(-1) BAP resulted in increment of hydrogen bonded polysaccharide and proteins concentrations, lipid structure and membrane fluidity. Treatments of 2.0 and 4.0 mg l(-1) BAP increased the existence of ferulic acid which also indicates the linkage of xylan and both cellulose and hemicellulose content. There was a significant decrease in the area of the peak which indicated the C=O stretching of proteins and pectins for 0.5 and 1.0 mg l(-1) BAP while there was a significant increase for 2.0 and 4.0 mg l(-1) of BAP treatments. The evaluation of molecular data from FT-IR spectroscopy as in the current study might help to estimate different physiological changes at the molecular level in plants depending on plant growth regulator and/or fertilizer applications. Thus, this FT-IR based method can be applied to in vitro propagation studies and also to studies focused on effects of pesticide and/or herbicide applications on plants to understand the metabolic changes in target species.