Plant immune activation in response to disease-causing pathogens entails proper spatiotemporal regulation of plant hormones and defense-activating metabolites. In Arabidopsis thaliana and numerous plant species, disease resistance to biotrophic and hemibiotrophic pathogens is underpinned by accumulation of these important immune signals, which include salicylic acid (SA), pipecolic acid (Pip), and N-hydroxypipecolic acid (NHP). SA, Pip, and NHP are essential for activating plant defense against various pathogens in local infected tissues, as well as priming distal, uninfected plant tissues in a phenomenon called systemic acquired resistance (SAR). Therefore, accurate quantification of these defense molecules is required to fully investigate the detailed regulation and function of plant immune networks. Here we present a highly sensitive and simple procedure to simultaneously quantify these immune-activating metabolites using state-of-the-art, high-performance liquid chromatographyChromatography coupled with tandem mass spectrometryTandem mass spectrometry (HPLC-MS). A detailed pipeline is presented, encompassing proper plant growth, pathogen infection, metabolite extraction, metabolite quantification, and data analyses. Overall, our current protocol enables accurate measurements of these molecules at low, physiologically relevant concentrations in plant tissues.