Downy thorn-apple Datura innoxia (Solanaceae) is an invasive plant species which can be introduced either accidentally or deliberately and have the ability to acclimatize in new environmental conditions. Scopolamine is a natural alkaloid which occur in several Datura species, including Datura innoxia. Occurrence of alkaloid in all plant organs is crucially important from the viewpoint of medical use, but also as a risk of toxicity for humans and animals, this paper presents the influence of alcoholic solvents on the separation ability of tropane alkaloids using a conventional extraction method (Soxhlet extraction) in order to investigate the content of scopolamine from Datura innoxia dry biomass from Romania at the maturity stage. Two solvents were selected, investigated and compared, including ethanol (96%) and 1-butanol (99.6%). The results showed that 1-butanol was most suitable for the extraction of scopolamine from Datura innoxia areal parts (leaves, flowers, seeds, stem and root) then ethanol because of the high degree of lipophilicity of this alkaloid. The quantitative analysis was performed using UV–VIS spectrophotometry technique. The calibration curve for the analyte under the optimum conditions was obtained with a proper correlation coefficient of 0.9930. Scopolamine was identified in all vegetative organs with higher concentration in 1-butanol and the total content (µg g−1) was 186.87 in leaves, 150.89 in flowers, 63.27 in seeds, 42.50 in stem and 58.10 in root. These studies provide new insights into the potential use of invasive plant species Datura innoxia for extraction of the content of tropane alkaloids, especially scopolamine using different solvents regarding the toxicity and therapeutic potential of this plant alkaloid.