Varicella-zoster virus (VZV) is a highly infectious agent that causes varicella and herpes zoster (HZ), which may be associated with severe neuralgia. Vaccination is the most effective way to reduce the burden of the diseases. VZV glycoprotein E (gE) is the major and most immunogenic membrane protein that plays important roles in vaccine efficacy. A quantitative assay for gE content is desirable for the VZV vaccine process monitoring and product analysis. In this study, 70 monoclonal antibodies (mAbs) were obtained after immunizing mice with purified recombinant gE (rgE). The collection of mAbs was well-characterized, and a pair of high-affinity neutralization antibodies (capture mAb 4A2 and detection mAb 4H10) was selected to establish a specific and sensitive sandwich enzyme-linked immunosorbent assay (ELISA) to quantify the native and recombinant gE. The detection limit of this assay was found to be 1.95 ng/mL. Furthermore, a reasonably good correlation between the gE content (as measured by the mAb-based quantitative ELISA) and the virus titer (as measured by the "gold standard" plaque assay) was observed when both assays were performed for tracking the kinetics of virus growth during cell culture. A total of 16 batches of lyophilized VZV vaccine were tested using the newly developed quantitative ELISA and classical plaque assay, demonstrating reasonably good correlation between gE content and virus titer. Therefore, this mAb-based gE quantitative assay serves as a rapid, stable, and sensitive method for monitoring viral antigen content, one additional quantitative method for VZV vaccine process and product characterization. This quantitative ELISA may also serve as a complementary method for virus titering.