A simple, sensitive, and reproducible high-performance thin-layer chromatographic method has been established for simultaneous analysis of trigonelline and 4-hydroxyisoleucine from fenugreek seeds ( Trigonella foenum-graceum ). Chromatography was performed on silica gel GF 254 with n -butanol-methanol-acetic acid-water 4:1.5:1:1 (υ/υ) as mobile phase. Densitometric scanning of the plates at 266 nm was used for analysis of trigonelline, and plates were scanned at 395 nm after derivatisation with ninhydrin reagent for analysis of 4-hydroxyisoleucine. The method was validated for specificity, precision (intraday and interday), accuracy, and robustness. Response was a linear function of the concentration of standard solutions in the range 100 to 1000 ng per band and 50 to 500 ng per band, respectively, with correlation coefficients of 0.9992 š 0.04 and 0.9986 š 0.06, for trigonelline and 4-hydroxyisoleucine. The accuracy of the method was checked by determination of recovery at three different levels; average percentage recovery was 99.39 š 0.34 for trigonelline and 99.03 š 0.26 for 4-hydroxyisoleucine. The method was found to be simple, precise, specific, sensitive, and accurate and can be used for routine quality control of fenugreek seeds and also for quantitative analysis of these marker compounds in processed fenugreek and its marketed preparations.