The present study was conducted to carry out the phytochemical investigation of Tanacetum gracile Hook. f. & Thomson and to develop a method for the simultaneous quantification of the isolated compounds in the extracts of T. gracile growing in different locations. Cluster analysis rectangular similarity matrix was performed to understand the chemical fingerprinting variations in the extracts. High-performance liquid chromatography–electrospray ionization-quadrupole-time-of-flight-mass spectrometry (HPLC–ESI-QTOF-MS) was used to quantify four bioactive compounds, and separation of the compounds was achieved on a reverse-phase C8 column using a mobile phase of acetonitrile: 0.1% formic acid in water with a gradient elution by maintaining the flow rate of 300 μL/min. The QTOF–MS was operated using the electro-spray ionization technique with the positive ion polarity mode. The calibration curves of four marker compounds were linear over the concentration range of 3.12–100 ng/µL ( R 2> 0.996). A specific, accurate and precise HPLC–ESI-QTOF-MS method was optimized for the determination of kaempferol, ketoplenolide, tetramethoxyflavone and artemetin both individually and simultaneously. Quantification of these chemical markers in different extracts was carried out using this validated method. Kaempferol was isolated for the first time from T. gracile.