In the current study, a novel method for high-throughput and sensitive determination of 12 phytohormones in plants was developed by using sequential solvent induced phase transition extraction (SIPTE) coupled with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). In sequential SIPTE, 0.1% formic acid (v/v) and 50mM NaHCO3 aqueous solution were used for enrichment and purification of alkaline and acidic phytohormones from the acetonitrile extract of plant tissues in sequence, in which hydrophobic solvent (toluene) was added to the acetonitrile aqueous mixture for driving the phase separation. Under optimized sequential SIPTE conditions, the phytohormones in acetonitrile extract of plant tissues could be effectively enriched and purified, which was in favor of the following UPLC-MS/MS analysis with less matrix effect. The phytohormones could be detected using the developed sequential SIPTE-UPLC-MS/MS method with the limits of the detection (LODs) ranging from 0.56 to 438.60pgmL(-1) and linear range over 2 orders of magnitude with correlation coefficients (r)>0.9970. The relative recoveries of the detected phytohormones were in the range of 85.1-114.6%. Finally, the proposed method was applied to simultaneous determination of endogenous phytohormones in different tissues of model plants (Oryza sativa and Arabidopsis thaliana) with small amount of sample size (5mg, fresh weight). The proposed method may be suitable for studying the distribution of phytohormones in model plants.