Background/Objectives: Potency is a critical quality attribute for vaccine development as well as clinical drug product (DP) lot release and stability testing. Animal studies have the potential to offer conclusive insights about the potency of vaccines by demonstrating technical relevance with respect to the hypothesized vaccine mode of action. However, animal studies are expensive, time-consuming, labor intensive, and, most importantly, involve the use of animals. Therefore, alternative in vitro potency assays should be explored. Methods: In this study, female BALB/c mice were immunized intramuscularly with various doses of a respiratory syncytial virus (RSV) mRNA vaccine V171 lots at day 0 and day 21. Vaccine-elicited immune responses were determined by ELISA (post-dose 1) and neutralizing assay (post-dose 2). These vaccine lots were also tested in a cell-based relative potency assay in which the ability of each lot to express the RSV F protein in Hep G2 cells was measured against a reference standard. Results: Effective Dose 50s (ED50s) of the vaccine lots were determined with probit models based on dichotomized ELISA or neutralizing titers. Statistical analysis demonstrated that the post-dose 2 neutralizing ED50 correlates with cell-based relative potency (Pearson’s correlation test ln (RP) and ln (ED50): correlation coefficient = −0.82; p-value = 0.047). Conclusions: These data merit the use of a cell-based potency assay to replace the animal study to support V171 vaccine development and to use for DP lot release and stability testing. This study also establishes proof-of-concept of using cell-based potency assays as an alternative to animal immunogenicity studies for mRNA-based vaccines.