Phytohormones are low molecular weight compounds that are produced by plants to regulate growth and development and also in response to biotic and abiotic stresses. The quantitative analysis of these molecules, which is essential for a better understanding of their physiological functions, is still particularly challenging due to their very low abundance in plant tissues. In this chapter, a rapid, sensitive, and accurate method for the quantification of acidic plant hormones is described. A fast and simple extraction procedure without purification or derivatization was devised, followed by optimized ultrahigh pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. The analytical procedure was validated in terms of selectivity, sensitivity, linearity, precision, recovery, and matrix effects. This protocol facilitates the high-throughput analysis of the main plant hormones and is applicable as a routine tool for a wide range of research fields such as plant-pathogen interactions, mutant screening, or plant development.