<p><i>Artemisia absinthium</i> Linn. (Family: Asteraceae), commonly known as Wormwood, is traditionally used against loss of appetite and mild dyspepsia. Its constituents viz., santonin (sesquiterpene lactone) and α- and β-thujone (monoterpene ketones) exhibit pharmacologic effects such as anthelmintic, neuro-stimulant, antioxidant, etc. They are also reported to exhibit potential toxicity on high or prolonged exposure and thus warrant accurate estimation. The current study aimed to develop a facile and precise method for the simultaneous estimation of santonin and thujones in the aerial parts of <i>A. absinthium</i> using high-performance thin-layer chromatography. Ascending development on aluminum-backed TLC plates pre-coated with silica gel 60 F₂₅₄ with toluene–acetone–glacial acetic acid (85:13:2, v/v) resulted in good resolution of bands. Their content was determined by densitometric scanning of the plates at 245&#xa0;nm. The method was validated as per the International Council for Harmonisation guidelines. The method demonstrated satisfactory linearity in the range of 400–2000&#xa0;ng/band for santonin (<i>r</i>² 0.9971) and 200–1000&#xa0;ng/band for α- and β-thujones (<i>r</i>² 0.9977 and 0.9995). LOD was calculated to be 129.1, 57.4, and 59.9&#xa0;ng/band and LOQ was estimated to be 391.4, 173.9, and 181.5&#xa0;ng/band for santonin, α- and β-thujone, respectively. The method demonstrated good recovery, reproducibility, and precision for the analytes. The content of santonin in <i>A. absinthium</i> was found to be 3.06 ± 0.42 mg/g while total thujones was found to be 0.57 ± 0.11 mg/g on a dry weight basis. The method was precise and robust for the simultaneous estimation of santonin, α- and β-thujone. The method can be applied for the quality control of <i>A. absinthium</i> preparations ensuring their safe use<i>.</i></p> Graphical abstract <p></p>