Background: Oldenlandia umbellata L. (Indian madder) is an antique Ayurvedic Indian herb and a source of various anthraquinone derivatives. The red dye from its roots has been used in diverse applications since ancient times. Objectives: To establish reliable and effective protocols for in vitro flowering of O. umbellata. Materials and methods: For in vitro flowering, organogenic calli were subcultured onto Murashige and Skoog (MS) medium supplemented with various concentrations of Naphthalene acetic acid (NAA) (0.15–1.0 mg/l) and Benzyladenine(BA) (0.5–1.5 mg/l) with and without 0.4% of coconut milk (CM). Results: The highest number of in vitro flowers (22.8%) and best response (92.73%) was achieved on MS medium supplemented with 0.7 mg/l NAA + 1.5 mg/l BA with 0.4% CM. It was found that MS medium devoid of BA promoted best root development (47.3 per calli) as well as response (100%). It was also observed that when embryogenic calli grown in depletion of required nutrition transferred to fresh media induced more flowering. In vivo and in vitro floral comparative analysis revealed that in vitro flower induction was required for short time duration (20.67 days) than in vivo flower. Conclusion: To the best of our knowledge, this is the first report on in vitro flowering and this study will help to overcome problems associated with flower development and seed production. As a result, this study may be a potent conservation tool to restore innate population size in its natural habitat. © 2017 Transdisciplinary University, Bangalore and World Ayurveda Foundation