The study's main goal is to test gastro retentive mucoadhesive Acyclovir (ACR) microspheres for various in vivo characteristics as a follow-up to the work done by Naresh et al (2022). The optimized ACR Azadirachta indica mucoadhesive microspheres (AAMM) were evaluated in vitro using a variety of parameters. Based on previous research findings, the authors used formula-8 (AAMM-8) for in vivo testing. The availability of plasma drugs was tested on six healthy rabbits of both sexes (weighing 2.-2.5kg). By using a parallel design, they were haphazardly parted into two groups of similar size. Later, the study involved HPLC analysis of the drug in the plasma, which required the fabrication of a spiked plasma sample and the development of an analytical procedure (a simple and cost-effective method for ACR).Pharmacokinetic parameters were measured in plasma samples. The study revealed that in vivo animal studies in rabbits showed good levels of ACR. After a single dose of formulation AAMM-8 (8.85mg/kg), the symmetrical mean Cmax values of formulation AAMM-8 (24.36±0.84µg/mL of ACR), were higher than those of ACR. The tmax values of the AAMM-8 were found to be 6 h. The AUC (0-10h) values were observed to be 176.0µg.h/mL, whereas (AUC)0-∞ was observed to be 248.5µg. h/mL. The AUMC readings were observed to be 724.62µg.h/mL. According to the findings, ACR reached systemic circulation faster than pure medication and had acceptable kinetic values.