Messenger RNA (mRNA) vaccines are highly effective against infectious diseases, but viral diversity has underscored the necessity for multivalent and combination vaccines. We hypothesize there could be interference of target protein translation in multivalent mRNA vaccines in cell-based assays used for potency testing. Following transfection into human cells, we observed that antigen protein translation is inhibited in multivalent formulations compared to their dose-matched monovalent counterparts using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Inhibited expression was further validated using Western blot and intra-cellular immunostaining with type-specific antibodies. Beyond direct antigen-specific analysis, LC-MS/MS proteomics data from the same assay without additional sample preparation provided a broad view of host cell responses, revealing proteome-level changes indicative of impaired global translation capacity in cells transfected with high dose mRNA(s). To the best of our knowledge, we report for the first time in literature that antagonistic interactions among mRNA constructs can reduce protein expression in multivalent drug products. Our study also demonstrates that LC-MS/MS is a viable alternative for potency testing without dependence on antibodies. These findings highlight the need for careful cell-based assay design, multivalent drug product reference standards for potency assessment, and deliberate mRNA dose optimization in multivalent formulations.