Global agricultural productivity is currently challenged by a range of emerging environmental stressors associated with the ongoing climate change scenario. The future of food security is critically reliant on the capacity to mitigate and manage these stresses on crops and trees. Over recent decades, substantial progress has been made in elucidating plant responses to various stresses, including heat, drought, and soil salinity, and in understanding how plant acclimatization to these responses is regulated by a limited number of signalling molecules, known as phytohormones. In response to abiotic stresses, abscisic acid (ABA) plays a pivotal role as it regulates, among other physiological processes, stomatal aperture and, consequently, the gas exchange and water uptake of plants. Therefore, methods for the comprehensive monitoring of changes in ABA levels are of particular significance. In this chapter, we provide a detailed description of a highly sensitive and accurate method for the quantitative analysis in plants using gas chromatography-coupled tandem mass spectrometry (GC-MS/MS). Additionally, we describe an indirect method for estimating ABA contents in Arabidopsis thaliana using ABA signalling reporter lines.