Plants encounter a variety of insect herbivores and have developed complex defense mechanisms against different feeding strategies. Phytohormones jasmonic acid (JA) and salicylic acid (SA) play a central role in recognizing and mounting appropriate defense response against pathogens. Primarily, JA provides resistance against chewing insect herbivores, whereas SA bolster defense against biotrophs and phloem-feeding insects (aphids and whitefly). Complex interaction of JA and SA signaling creates a plant stress memory which helps to generate elevated defense response to future insect threats. Phytohormone quantification can provide significant insights about activated plant defense response and involved signaling pathways. Here, we describe a detailed and reproducible protocol for phytohormone quantification (SA, ABA, JA, and JA-Ile) in insect-fed Arabidopsis leaves tissues. JA and JA-Ile quantification results clearly indicate their contribution in plant adaptation and defense response.