One of the hottest topics in plant hormone biology is crosstalk, that is, multiple classes of plant hormones interact with each other through signaling networks. To better understand these interactions and their precise roles in the complex regulating network, it is significant to investigate the spatial and temporal distribution of multiple plant hormones even all phytohormone classes simultaneously from one piece of plant tissue sample. In this study, we develop a high-sensitivity and high-throughput method for the simultaneous quantitative analysis of 44 phytohormone compounds, covering all of 10 major classes of phytohormones (including Strigolactones, brassinosteroids, gibberellins, auxin, abscisic acid, jasmonic acid, salicylic acid, cytokinins, ethylene and polypeptide hormone (phytosulfokine)) that are currently known, from only one hundred milligram of plant sample. These compounds were grouped and purified separately with a tailored solid phase extraction procedure based on their physicochemical properties and then analyzed by LC-MS/MS. The recoveries ranged from 49.6% to 99.9% and the matrix effects from 61.8-102.5%, indicating a good purification result of the overall sample pretreatment design. LOQs ranged from 0.06 to 1.29 pg/100 mg FW and the precision less than 13.4%, indicating high sensitivity and good reproducibility of the method. The applications in different plant matrixes demonstrated its wide applicability. These advantages will make the method helpful in clarifying the crosstalk networks of phytohormones.