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This paper describes a method to detect and quantitate the endogenous plant hormones (±)-2-cis-4-trans-abscisic acid, (−)-jasmonic acid, and salicylic acid by means of ultrahigh-performance liquid chromatography−tandem mass spectrometry (UHPLC−MS/MS) in hybrid rose leaf matrices. Deuterium-labeled [2H6] (+)-2-cis-4-trans-abscisic acid, [2H6] (±)-jasmonic acid, and [2H4]-salicylic acid were used as internal standards. Rose samples (10 mg) were extracted with methanol/water/acetic acid (10:89:1) and subsequently purified on an Oasis MCX 1 cm3 Vac SPE cartridge. Performance characteristics were validated according to Commission Decision 2002/657/EC. Recovery, repeatability, and within-laboratory reproducibility were acceptable for all phytohormones tested at three different concentrations. The decision limit and detection capability for (±)-2-cis-4-transabscisic acid, (−)-jasmonic acid, and salicylic acid were 0.0075 and 0.015 μg/g, 0.00015 and 0.00030 μg/g, and 0.0089 and 0.018 μg/g, respectively. Matrix effects (signal suppression or enhancement) appeared to be high for all substances considered, implying the need for quantitation based on matrix-matched calibration curves.