The possibilities in the identification and quantitation of the constituents of Rubia tinctorum L.'s root, called also madder root, was described and compared by gas chromatography (GC)-MS, high-performance liquid chromatography (HPLC)-UV/photodiode array detection (DAD) and HPLC-MS: chromatographic analyses were carried out in parallel, from the same samples/extracts/hydrolyzates. Anthraquinone glycosides, anthraquinones, carboxylic acids and sugars were determined directly in the presence of the matrix and in its extracts without and subsequently to hydrolyses. Hydrolyses were performed as a function of time, with hydrochloric and trifluoroacetic acids, as well as enzymatically. Data revealed that as hydrolyzing agent trifluoroacetic acid is to be preferred. Madder root's anthraquinones (pseudopurpurin/purpurin, alizarin, lucidin, munjistin, nordamnacanthal) were identified on the basis of their absorption spectra (HPLC-DAD) and fragmentation patterns by GC-MS and HPLC-MS, equally. Reproducibility of anthraquinone's quantitation, by HPLC-DAD and GC-MS, in the concentration ranges of 4 x 10(-5) to 3 x 10(-2)g/g dried sample, provided an average reproducibility of 4.2% (varying between 0.9 and 9.4% relative standard deviation (RSD percentages)). Carboxylic acids (malic, citric, quinic, rosmarinic acids) and saccharides (xylose, ribose, fructose, glucose, sucrose, primverose) were quantified as their trimethylsilyl (oxime) ether/ester derivatives by GC-MS, in the concentration ranges of 10(-5)g to 10(-2)g/g dried sample, with an average reproducibility of 4.7% RSD.