Acyclovir (ACV) is a synthetic acyclic nucleoside analogue active against herpes simplex virus type 1 and 2 (HSV-1 and HSV-2). The current research entails optimization, development, and validation of the sensitive, accurate, and precise high performance liquid chromatography-photo-diode array detector (HPLC-PDA) bioanalytical method for quantification of ACV in rat plasma. The central composite design (CCD) of Design Expert (quality by design tool) was employed for identification of significant attributes (flow rate and concentration of buffer), which affected the performance of the developed method. The elution of ACV was achieved by separating the XBridge C18 column and the mobile phase comprising of the potassium dihydrogen phosphate buffer (pH-6.8) and acetonitrile in a 90:10 v/v ratio pumped at a flow rate of 1.0 mL/ min. The method was validated as per International Council for Harmonization (ICH) guidelines in terms of selectivity, linearity, recovery, accuracy, and precision. The values of the lower limit of detection and the lower limit of quantification were found to be 30 and 100 ng/mL, respectively. Conclusively, the study showed superior performance with high robustness, sensitivity, and specificity of the developed bioanalytical method. The developed quantification method was applied for estimating pharmacokinetic (PK) parameters of ACV loaded vesicular systems (ethosomes, elastic liposomes, colloidal solution, and solution) transdermally applied to rat skin (using a previously published report). The method was successful in quantifying PK profiles for comparative assessment with a high robustness, re-validity, re-transferable, and simplicity approach.