Herbal formulation is one of the novel drug delivery systems which possesses various advantages, including increasing drug solubility, enhancing dissolution rate, bioavailability, etc. Piper betle leaves Linn. (Family: Piperaceae) have been used in Chinese and Indian folk medicine for centuries, and recently been proposed to be used as a chemoprevention agent because of its anti-oxidant activity. The cytotoxicity of the aqueous extract of PBL was carried out to find its anticancer properties. A 3-level full factorial design was employed to optimize the process conditions for formulating spherical shaped microspheres with higher encapsulation of drug. Formulation A1 to A9, B1 to B9 and C1 to C9 were prepared using three independent variables (drug-polymer ratio and stirring speed) and evaluated for dependent variables (shape, percentage yield, particle size, percentage drug loading and percentage cumulative release of drug in 24 h). The cytotoxicity studies on Hep-2 cell line of sodium alginate microspheres exhibited a mean CTC50 of 95.20 µg/ml. In case of BSA and chitosan microspheres this was found to be 90.00 and 94.00 µg/ml respectively.