INTRODUCTION: Madder (Rubia tinctorum) has been used since ancient times as a source of pigments for dyeing and painting. Madder dyes are localised in roots and the native chemical population is composed of glycosiled and aglycone compounds. The aim of this study is to elaborate an efficient extraction process without any chemical denaturation of dyes. OBJECTIVE: To compare an optimised ultrasonic process, using for madder dye extraction, with two conventional procedures and to determine the efficiency of ultrasound on these vegetable matrix. METHODOLOGY: Madder roots were extract in a methanol-water mixture in 37 : 63 (v/v) for ultrasound and 80 : 20 (v/v) for reflux and agitation. HPLC-PAD analyses showed the anthraquinone proportion for each extraction process and their denaturing effects. Finally, cytohistological observations were made to show the consequence of each process on the cell organisation in madder roots. RESULTS: The results showed that the amount of extracted dyes was higher with UAE than with agitation and reflux. HPLC-PAD analysis revealed that the anthraquinone composition differed according to the extraction procedure. The UAE extracts presented an important richness in terms of anthraquinonic compounds that suggests a preserving effect. Cytohistological observations showed that the main alterations concerned the cell walls of phloem. After UAE the walls exhibited numerous pitted areas reflecting an ultrasound-induced cavitation that enhances the extraction effectiveness of this method. CONCLUSION: The study has shown the improvement of madder roots extraction both quantitatively and qualitatively using the efficiency of ultrasound-assisted extraction in comparison with magnetic agitation and reflux techniques