Amsonia orientalis (European Bluestar) is a critically endangered plant species with medicinal and ornamental properties. The rare availability of the species in nature limits its potential to be used for various purposes. However, plant tissue culture is an effective method for the cultivation of such vulnerable species without damaging their natural populations, which are very limited in nature for scientific purposes. By taking advantage of plant tissue culture, this study aimed to measure the phenolic substance and flavonoid contents in leaf extracts of in vitro-propagated Amsonia orientalis, and to investigate their antioxidant potentials through phosphomolybdate and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays. The crude extracts prepared in water, aqueous ethanol, methanol, and acetone were tested. The highest phenolic substance content was found in the ethanolic extracts, while statistically the same flavonoid contents were found in the ethanolic, methanolic, and acetone extracts. Although the water extract had lesser flavonoid content, it exhibited a notable antioxidant property. The ethanolic leaf extract gave the highest antioxidant and DPPH radical scavenging activity, especially when used at 1 mg mL(-1) concentration. Also, the TLC fingerprint profile validated the presence of valuable phytoconstituents in the leaves of the plant. This study indicated that ultrasound-assisted extraction of minimal amounts of dried leaf samples from in vitro-propagated plants might be adequate for the pre-screening of the antioxidant capacity of rare plant species.