A thin-layer chromatography method for the separation and in situ densitometric quantitation of sanguinarine and berberine was validated, following selected guidelines from the International Council for Harmonisation. The method was sensitive to both alkaloids and quantities < 1.5 ng/spot could be detected and quantified with precision, accuracy, and robustness. It required minimal amounts of plant tissue for sample processing. Due to its simple execution, up to 20 analyses could be performed from beginning to end in less than 2 h.