In the present study, mucoadhesive alginate microspheres of acyclovir were prepared to prolong the gastric residence time using a simple emulsification phase separation technique. The particle size of drug-loaded formulations was measured by SEM and the particle size distribution was determined using an optical microscope and mastersizer. The release profile of acyclovir from microspheres was examined in simulated gastric fluid (SGF pH 1.2). The particles were found to be discreet and spherical with the maximum particles of an average size (70.60 ± 2.44 µm). The results indicated that the mean particle size of the microspheres increased with an increase in the concentration of polymer and decreased with increase in stirring speed. The entrapment efficiency was found to be in the range of 51.42-80.46%. The concentration of the calcium chloride (% w/v) of 10% and drug-polymer ratio of 1:4 resulted in an increase in the entrapment efficiency and the extent of drug release. The optimized alginate microspheres were found to possess good mucoadhesion (66.42 ± 1.01%). The best fit model with the highest regression coefficient values (R²) was predicted by Peppas model (0.9813). In Gamma scintigraphy analysis, the section of GIT was critically analyzed and much differentiation was present at each time point after oral administration, which revealed that the optimized formulation demonstrated gastroretention in vivo for more than 4 h, which revealed that optimized formulation could be a good choice for gastroretentive systems.