The immune system of our body plays an important role in maintaining the physiological activities of our body by protecting it from various foreign microorganisms and pathogens. Some recent studies reveals that the exploration of plant obtained from natural products having immunomodulatory potential have gained therapeutic significance. T-cell proliferation assay is one of the reliable in vitro methods by which the immunomodulation of leaves can be determined. This chapter focuses on the isolation and preparation of cytotoxic T cells from murine spleen; preparation of processed extracts; cell culture and stimulation conditions with mitogens; and quantification with colorimetric and fluorescence-based assays (ELISA). It also discusses key aspects such as optimization of extraction concentration of natural products, cytotoxicity control, and selection of adequate mitogens (Concanavalin A) to achieve reliable and reproducible results.