The objective of this study was selection of most influential variable for the preparation of gastro adhesive nanoparticles of acyclovir. Effect of formulation and processing variables on various response variables were studied by a Taguchi standard orthogonal array L-8 design. Independent variables studied were the amount of gelatin, amount of glutaraldehyde, amount of Pluronic F-68, acetone addition rate, pH, stirring time and stirring speed. The size of all nanoparticulate formulations prepared as per the experimental design (Taguchi screening design) varied between 165 and 1610 nm, PDI between 0.360 and 1.00, Q6 between 7.31-34.93%, T60% between 19.2-37.6 h, entrapment efficiency between 15.70 and 83.12%, loading efficiency between 39.72 and 80.49% and mucoadhesive strength between 3.959-11.02 g. Pareto ranking analyses showed that the two most important factors affecting the selected responses were amount of gelatin and amount of Pluronic F-68 (p<0.05).