An HPLC method with postcolumn derivatization was developed and validated for the determination of theanine content in tea dietary ingredients and supplements. A variety of common commercially available supplement forms such as powders, liquid tinctures, tablets, softgels, and gelcaps, as well as three National Institute of Standards and Technology Camellia sinensis Standard Reference Materials were investigated in the study. A simple extraction procedure using citrate buffer at pH 2.2 allowed for the analysis of theanine without additional cleanup or concentration steps, even at low ppm levels. Theanine was separated from other naturally occurring amino acids using a cation-exchange column and detected using a UV-Vis detector after derivatization with ninhydrin reagent. A single-laboratory validation demonstrated that specificity, accuracy, precision, and other method performance parameters have met the requirements set for theanine analysis by the AOAC Stakeholder Panel on Dietary Supplements.