© American Society for Clinical Pathology, 2018. All rights reserved. Background: The therapeutic efficacy and safety of stored red blood cells (RBCs) relies on minimal in-bag hemolysis. The accuracy of current methods of measuring hemolysis can suffer as a result of specimen collection and processing artefacts. Objective: To test whether Raman spectroscopy could be used to assess hemolysis. Methods: RBCs were stored for as long as 42 days. Raman spectra of RBCs were measured before and after washing, and hemolysis was measured in supernatant by visible spectroscopy. Results: Raman spectra indicated increased concentrations of oxyhemoglobin (oxyHb) and methemoglobin (metHb), and decreased membrane fluidity with storage age. Changes in oxyHb and metHb were associated with the intraerythrocytic and extracellular fractions, respectively. Hemolysis increased in a storage age-dependent manner. Changes in Raman bands reflective of oxyHb, metHb, and RBC membranes correlated with hemolysis; the most statistically significant change was an increased intensity of metHb and decreased membrane fluidity. Conclusions: These data suggest that Raman spectroscopy may offer a new label-free modality to assess RBC hemolysis during cold storage.