Resurgence of vitamin D deficiencies in recent years has ascribed the need for expansion of fortification strategies in food. Alluding to the fat soluble and sensitive chemical nature of vitamin D, existing fortification strategies invariably require using a substantial amount of fat as carriers for vitamin D. Though milk proteins have demonstrated good binding properties with vitamin D; allergen issues, lactose intolerance, and the need to cater to vegan population deter its extensive use. In this study, soluble leaf proteins extracted from low-alkaloid tobacco leaves were investigated as a possible carrier. Crude tobacco leaf proteins were extracted by a high-throughput mechanistic process, followed by a freeze-drying process to encapsulate vitamin D. Up to 84.68% (w/w) of vitamin D was successfully retained by tobacco leaf proteins using the process developed, indicating that crude leaf protein recovered from tobacco could be employed as an effective carrier for vitamin D.