<jats:title>Abstract</jats:title><jats:p>In the experiment, a high‐performance liquid chromatography and electrospray ionization‐tandem mass spectrometry with selected reaction monitoring was used to simultaneously determine various classes of phytohormones, including indole‐3‐acetic acid, α‐naphthaleneacetic acid, 2‐chlorobenzoic acid, 4‐chlorobenzoic acid, indole‐3‐butyric acid, gibberellic acid, 2,4‐dichlorophenoxyacetic acid, 2‐naphthoxyacetic acid, abscisic acid, 2,3,5‐triiodobenzoic acid, uniconazole, paclobutrazol and 2,4‐epibassinolide in rape tissues. The analyses were separated by an HPLC equipped with a reversed‐phase column using a binary solvent system composed of methanol and water, both containing 0.1% of formic acid. The matrix effect was also considered and determined. The technology was applied to analyze rape tissues, including roots, stems, leaves, flowers, immature pods and rape seeds. The rape tissues were subjected to ultrasound‐assisted extraction and purified by dispersive solid‐phase extraction, and then transferred into the liquid chromatography system. The detection limit for each plant hormone was defined by the ratio of signal/background noise (<jats:italic>S</jats:italic>/<jats:italic>N</jats:italic>) of 3. The results showed perfect linearity (<jats:italic>R</jats:italic><jats:sup>2</jats:sup> values of 0.9987–1.0000) and reproducibility of elution times (relative standard deviations, RSDs,<1%) and peak areas (RSDs,<7%) for all target compounds.