Abstract: The quantitative analysis of isoquinoline alkaloids in Chelidoninemajus L. was investigated and a HPTLC method has been established. Using this method,we separated and determined eight isoquinoline alkaloids, i. e. chelidonine, protopine,berberine, coptisine, tetrahydrocoptisine, 6- methoxydihydrochelerythrine, 6- methoxy-dihydrosanguinarine and dihydrosanguinarine. The HPTLC method developed used onedeveloping system on high performance silica gel plate (10×10 cm). After separationof these eight alkaloids, fluorescence derivatization was carried out in situ. The content wasdetermined by fluorescence scanning. TLC fluorescence derivatization, fluorescenceenhancement and fluorescence stability have been studied. Experiments showed that thismethod is simple, fast, highly sensitive and highly selective.