Psychotria ipecacuanha, is a plant species with known medicinal properties that is critically endangered due to overexploitation of natural populations. Although the difficulties in conventional propagation by seed and by vegetative propagation are generally understood, the present study enhances our knowledge by describing efficient plant regeneration and root induction protocols for P. ipecacuanha while comparing alkaloid content (emetine and cephaeline) in in vitro-derived tissues. Stem node explants were cultured on MS medium MS supplemented with indolbutiric acid (IBA) in semi-solid media and the RITA® temporary immersion bioreactor. The highest root formation (81%) was in MS + 1.5 mg L−1 IBA in the bioreactor. After 24 months of acclimatization, the plants cultivated in MS + 0.50 and 1.0 mg L-1 of IBA had the highest number of roots (3), with mean values of 10.47 and 9.40 cm, respectively. The cultures coming from 1.0 mg L−1 and 0.5 mg L−1 IBA in the bioreactor contained higher cephaeline content, with a relative area of 14.2 and 14.9%, respectively. For emetine, the 1.0 mg L−1 IBA cultures in the bioreactor, 0.5 mg L−1 IBA and MS0 cultures contained higher content than the other treatments, with a relative area of 10.2, 10.2 and 10.1%, respectively.