The presence of anthraquinones gives many plants of the Rubiaceae commercial importance. The root of Oldenlandia umbellata L. (Indian madder) is a source of natural dyes. In the present study, successful protocols have been developed for rapid root development and dye production from in vitro raised normal roots. For rapid root development, organogenic calli were subcultured onto MS medium supplemented with various concentrations of NAA (1-naphthaleneacetic acid) (0.15-1.0. mg/l) with CM (coconut milk) (0.2-0.6%). The best root development (47.3 per calli) as well as response (100%) was obtained in the MS medium supplemented with 0.7. mg/l NAA and 0.4%CM. The dye obtained from in vitro raised normal roots was analyzed by thin-layer chromatography (TLC), high performance thin-layer chromatography (HPTLC), spectrophotometric quantification followed by high performance liquid chromatography (HPLC). The in vitro roots represented 338.84 μg/ml AE (alizarin equivalence) of anthraquinones, whereas the in vivo roots were found to possess 361 μg/ml AE. To the best of our knowledge, this is the first report on anthraquinone dye production from O. umbellata which enhances the production of fresh pigment and through in vitro culture allows harvest throughout the year. © 2011 Elsevier B.V.