This study aimed to investigate the effects of drought stress on Amsonia orientalis, an endangered ornamental plant with a limited natural distribution in Europe. Effects of polyethylene glycol (PEG)-mediated drought stress (-0.15, -0.49, -1.03 and -1.76 MPa osmotic potentials) were tested on in vitro cultures. In general, root lengths and numbers, total protein, chlorophyll a and carotenoid contents were negatively influenced at elevated levels of the stress factor. The successive decrease in the tested osmotic potentials resulted in gradually higher H2O2, malondialdehyde (MDA) and proline contents. Activities of the antioxidant enzymes, peroxidase (POD) and catalase (CAT), were found to be enhanced in response to the decreasing osmotic potential tested, whereas increased superoxide dismutase (SOD) activity was observed at the -0.15 MPa osmotic potential. Strong activation of POD enzymes under drought stress suggests that POD enzymes might have a major role in regulating the H2O2 content, while CAT has only a supplementary role in A. orientalis. These results indicated that although A. orientalis is susceptible to long-term drought, the species may survive during mild drought stress because the development of the plant was not totally inhibited but only limited. Nevertheless, the species should be introduced to well-irrigated lands, after evaluation of the soil's water status, in order to ensure the continuation of its generations.