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The review analyzes and summarizes the latest literature on the history of development and the current state of methodological approaches to the identification and quantification of phytohormones in plant tissues. Phytohormones play a key role in the regulation of physiological processes throughout the life cycle of plants – from seed germination to aging. In tissues of plants, their concentrations are very low (from 10-9 M to 10-6 M), so the development of fast, highly efficient, comprehensive, and at the same time reliable approaches to the determination of phytohormones is extremely urgent. The article gives a brief description of the main classes of phytohormones, describes their functional activity. The substantiated and checked sequence of procedures on the extraction of plant hormones, their separation from interfering substances, quantification methodology for indol-3-acetic, abscisic, gibberellic (GA3), salicylic acids and five cytokinins that combines a high-performance liquid chromatography with mass spectrometry are presented. The article describes four chromatographic methods of profiling, gives the conditions for detecting phytohormones of different classes and ionization of these substances in a mass spectrometer. The importance of phytohormones profiling in plant tissues for further applied in biotechnological approaches, in particular in metabolic engineering, for the formation of stress resistance in crops is discussed.