Theanine is a unique amino acid almost exclusively found in tea which is considered to have positive effects on human health. Data on theanine methods of analysis are quite rare in the literature and extraction parameters are not well documented. In this study, optimum extraction parameters such as particle size, extraction temperature and time, and derivatization time with coloring reagent were determined. High performance liquid chromatography (HPLC) with fluorescence detector was used for the determination of theanine, and chromatographic separations were achieved by using reverse-phase C(18) column. HPLC gradient program was improved to get better resolution of theanine. Linearity, selectivity, limit of detection (LOD), limit of quantification (LOQ) and precision of method (intra and inter-day) were also determined. Best extraction conditions for particle size, extraction temperature, extraction time and derivatization time were found to be 150-300 mu m, 80 degrees C, 25 min and 2 min, respectively. Precision of the HPLC method was very high since intra-day and inter-day variations were 1.14% and 0.96%, respectively. LOD and LOQ values were found as 20 ng/mL and 50 ng/mL indicating the method is highly sensitive. (C) 2011 Published by Elsevier Inc.