Natural vanilla is a popular flavouring essence which is in high demand globally. In order to meet the demand, superior vanilla plant varieties are needed for high quality vanilla pods production. Clonal propagation is a viable means of producing high yielding vanilla plantlets. However, the production of vanilla plantlets through the tissue culture route is still very low. We have developed an efficient regeneration protocol by using cytokinin as the sole plant growth regulator to clonal propagate the plantlets from the root tips of Vanilla planifolia (V. planifolia) Andrews. The key steps involved in the regeneration protocol involve induction of callus from the excised root tips followed by the conversion to PLBs before eventual formation of shoot primordia in the MS media supplemented with 1 mg/L BAP. We use isobaric tags for relative and absolute quantitation (iTRAQ) proteomic approach to investigate the underlying molecular and cellular mechanisms involved in the induction of callus (VR-VC) and PLBs conversion (VC-VP). A total of 595 and 1237 unique proteins with abundance changes of > 1.5 or < 0.67 - fold were identified in VR-VC and VC-VP respectively. The results indicated that for the dedifferentiation of root tip into callus, the GO terms enriched in upregulated proteins were involved in carbohydrate metabolic process, oxidation-reduction and chloroplast while the down regulated proteins were associated with protein transport, protein binding and gene expression. For the differentiation of callus to protocorm, the up-regulated proteins involve thylakoid membrane, plastids and photosystem I while the down regulated proteins involve RNA binding and metabolic process. The findings provide insights into the steps involved in the tissue culture of vanilla orchids which will be useful in devising effective strategies for the micropropagation and breeding programme of V. planifolia.