<p>This study investigates the presence of anise (<i>Pimpinella anisum</i> L.) in three eighteenth-century pharmaceutical preparations, preserved in the collection of the National Museum in Prague. A chemotaxonomic approach was employed using complementary analytical techniques: HPLC–UV, comprehensive two-dimensional gas chromatography coupled with mass spectrometry (GC × GC–MS), and high-performance liquid chromatography with tandem mass spectrometry (HPLC–MS/MS). <i>Trans</i>-anethole, the principal aromatic constituent of anise, was quantified (or detected) in all historical samples as well as modern reference materials. GC × GC–MS enabled the identification of key markers of anise (<i>trans</i>-anethole, <i>p</i>-anisaldehyde, estragole, and γ-himachalene) in the historical samples. HPLC–MS/MS confirmed the presence of <i>trans</i>-pseudoisoeugenyl 2-methylbutyrate—a highly specific chemotaxonomic marker of anise—in one historical specimen. These results confirm the authenticity of all three preparations as genuine anise-based remedies and demonstrate the value of combined chromatographic techniques in the chemical analysis of historical pharmaceutical remains. In addition, the full ESI⁺-MS/MS spectrum of <i>trans</i>-pseudoisoeugenyl 2-methylbutyrate was recorded and structurally elucidated, providing novel reference data on this previously poorly characterized chemotaxonomic marker of anise.</p> Graphical abstract <p></p>