We developed reproducible protocols for micropropagation, callus culture, and root culture of the medicinal plant Phyllanthus urinaria, P. niruri, P. tenellus, P. corcovadensis, P. caroliniensis, P. stipulatus, and P. fraternus by using single node explants. Genotype-linked differences are visible among the Phyllanthus species concerning shoot culture, callus culture, and root culture growth. The protocols developed for phytochemical screening of callus and root extracts of P. urinaria, P. caroliniensis, P. stipulatus, and P. fraternus have shown the production of sterols and triterpenes. Both compounds are known to account for the antinociceptive activity of the methanolic extracts as glochidone and stigmasterol have strong activity against neurogenic and inflammatory pain. Similarly, methanolic callus extracts of P. tenellus, P. niruri and P. corcovadensis have potent analgesic properties, however phenolics are major compounds isolated from these species. The optimized micropropagation, callus culture, and root culture protocols offer the possibility to use cell/root culture techniques for vegetative propagation and secondary metabolite studies.