The present study evaluated a large number of cellulose, polyamide or silica gel TLC systems suggested for analysis of phenols but without success for separation of glyceollin I-III (GI-III, data now shown). However, these compounds are easily separated by using multiple developments of formamide-impregnated silica gel layers, a sorbent described for TLC of Umbelliferae drugs. Quantitation of GI-III in plant extracts is then achieved by using this system in place of the last purification-quantitation steps, followed by normal UV spectrometry of the eluted spots. This method, when combined with radioautography, may also find application in radiotracer studies of the biosynthesis of GI-III.