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Phytohormones act at relatively low concentrations as major regulatory factors of plant growth and development, and cross talk of phytohormones is currently of great interest throughout the plant science community. To meet this demand, a method that is capable of simultaneously analyzing diverse plant hormones is essential. This paper introduces a high-performance liquid chromatographic separation technique coupled with sensitive and selective ion trap mass spectrometry to simultaneously determine 24 or more acidic and alkaline phytohormones, including auxin, cis- and trans-abscisic acid, 11 cytokinins, and 10 gibberellins, in a single injection of sample. A binary solid-phase extraction using Oasis MCX cartridges for cations and Oasis MAX cartridges for anions was used to prepurify more than 24 acidic and alkaline phytohormones from a single plant extract. The method showed good linearity for all 24 phytohormones with R (2) values ranging from 0.9903 to 0.9997. Limits of detection for most of the phytohormones were in the femtomole range with some extending into the sub-femtomole range. This method was applied to hundreds of plant samples comprising different tissues from various plants, including herbaceous, woody climbing, and woody plants to demonstrate feasibility and to validate the methodology.