Premise of the StudyCurrent methods for quantifying herbivore‐induced alterations in plant biochemistry are often unusable by researchers due to practical constraints. We present a cost‐effective, high‐throughput protocol to quantify multiple biochemical responses from small plant tissue samples using spectrophotometric techniques.Methods and ResultsUsing Solanum lycopersicum and Medicago polymorpha leaves pre‐ and post‐herbivory, we demonstrate that our protocol quantifies common plant defense responses: peroxidase production, polyphenol oxidase production, reactive oxygen species production, total protein production, and trypsin‐like protease inhibition activity.ConclusionsCurrent protocols can require 500 mg of tissue, but our assays detect activity in less than 10 mg. Our protocol takes two people 6 h to run any of the assays on 300 samples in triplicate, or all of the assays on 20 samples. Our protocol enables researchers to plan complex experiments that compare local versus systemic plant responses, quantify environmental and genetic variation, and measure population‐level variation.