It is important to understand the molecular mechanisms underlying oxygen toxicity, which contributes to multiple human disorders. The archetype model of oxygen toxicity is neonatal lung injury induced by hyperoxia exposure. Here, we utilized plasmonically enhanced Raman spectroscopy (PERS) in combination with fluorescence and proteomic analysis to provide comprehensive information on hyperoxia-induced biomolecular modifications in neonatal mouse lung fibroblasts (nMLFs). During this study, we made the novel observation that hyperoxia induces intracellular acidification in nMLF, which we probed in real-time using label-free PERS. We found that intracellular acidification induces conformational modifications in proteins followed by significant changes in Raman vibrations corresponding to aromatic amino acids such as phenylalanine and tryptophan as well as cysteine moieties. Hyperoxia-induced intracellular pH changes and subsequent modifications in protein expression and associated post-translational modifications within the cells were further validated by fluorescence and proteomic analysis. These new insights may help identifying unique oxidant stress-induced mechanisms in disease processes and may guide the development of more efficient therapeutic strategies.