Croton bonplandianus is an important medicinal herb and has been used to prepare herbal formulations to cure many diseases. Its potential to heal wounds can be exploited as an effective formulation in medicines. In vitro-raised plantlets provide a suitable alternative to mass multiplication and cell culture-based metabolite production. It is an effective strategy for the conservation of plants in their natural and native habitats. An efficient protocol for in vitro shoot multiplication of Croton bonplandianus has been developed using internodal segments as explants. It was observed that shoot multiplication could be induced on a slightly modified Murashige and Skoog’s (MS) medium supplemented with 6-benzylaminopurine, Naphthalene Acetic Acid, Indole 3-Acetic Acid, in various combinations. Shoot proliferation and root induction were induced with an enhanced number of plantlets, and optimization was successfully done to obtain viable and healthy plantlets with a success rate of more than 82%. The optimized protocol can be used to optimize cell suspension cultures.