Cyclotides are an emerging class of disulfide-rich plant cyclic peptides, believed to be present in plants for defence purposes. Their exceptional thermal, chemical and enzymatic stabilities make them suitable as therapeutic agents, agrochemicals, molecular imaging probes and pharmaceutical scaffolds for drug delivery. Nearly 350 cyclotides have been identified and reported to date, while more than 150,000 diverse cyclotides are estimated to occur in plants. With the current chemical identification methodologies, cyclotides can be identified even from crude extracts. This review will extensively describe the latest strategies pertaining to the screening of plants for the presence of cyclotides. Although identification of cyclotides in plants will help to explore their distribution, evolution, diversity and biological activities, natural plant extraction is not a sustainable and reliable method of such plant metabolite production. This can be attributed to several reasons such as inconsistent and non- uniform supply due to geographical and climatic conditions. Moreover, extraction of metabolites from the non-native, rare and endangered plant is not sustainable. Therefore, in vitro production technologies independent of the natural source availability are being explored to facilitate commercial applications of cyclotides. Owing to the complex structure, the chemical synthesis and recombinant microorganism-based methods of production provide limited yields of cyclotides. Production of biopharmaceutical peptides in plant cell cultures is a safe and commercially applicable alternative which can overcome some of these challenges. Hence, strategies for sustainable production of cyclotides by plant in vitro systems is discussed in-depth in this review.