Polyphenolic compounds are abundantly found in natural dyes. The cupric reducing antioxidant capacity assay originally developed in our laboratories was utilized to estimate the total polyphenolic content of natural dyes (i.e., Rubia tinctorum L, Curcuma longa L., Alkanna tinctoria, Matricaria chamomilla, Dactylopius coccus Costa) for the first time. The polyphenolic compounds such as ellagic acid, gallic acid, quercetin, rutin, fisetin, myricetin, kaempferol, luteolin, apigenin, and morin were all capable of reducing the Cu(II)-neocuproine reagent to the Cu(I)-neocuproine chelate showing maximum absorbance at 450 nm, and the responses of synthetic mixture components were additive in accordance with Beer's law. As a comparative reference method, the AlCl3/potassium acetate spectrophotometric method was applied to total flavonoid assay of these dyes. The results of the proposed and reference methods were correlated with high performance liquid chromatography (HPLC) findings expressed in the units of quercetin (QR) equivalent polyphenolic concentration. The individual phenolic constituents of dye extracts were identified and quantified by HPLC on a C18 column (alizarin, curcumin, carminic acid, etc.). The method of standard additions was applied to the solutions of these dyes by adding standard increments of QR and measuring the resulting absorbances. (C) 2010 Elsevier B.V. All rights reserved.