By implementing supercritical fluid extraction techique to thirty different types of natural resources, a new systematic experimental routine consists of extraction, chromatographic analysis and bioassay of bioactive substances is developed. Also, by performing conventional organic liquid solvent extractions using chloroform and/or methanol to all the resources and by comparing the experimental results with those obtained by the supercritical CO2 extraction, a criterion of evaluation whether the supercritical fluid extraction process can be a viable alternative or not for separation process development is discussed. In addition, the optimum separation conditions of supercritical CO2 extraction of each prodrug substances from the plants are discussed. Expectations from the results obtained in the present study goes to the prime utility as a basic and reliable guide for the future development of supercritical fluid extraction process for new prodrug substances from natural products.