Extracellular tonicity has a significant influence on human red blood cell deformation capability. Advancements in the area of laser physics and optical trapping have opened up a plethora of applications for understanding cell structure and dynamics. Here, Raman Tweezers technique was employed to investigate the impact of extracellular tonicity by exposing human red blood cells to both hypertonic and hypotonic intravenous fluids. Heme aggregation was observed in hypertonic saline solution, accompanied with damage in membrane protein. Loss of intracellular hemoglobin in hypotonic solution was evident from the decrease in porphyrin breathing mode present at 752 cm-1. Oxygen binding to the central iron in the red blood cell heme was also affected under both hyper/hypo tonicity conditions. Morphological deviation of discocytes to echinocytes/spherocytes were also evident from quantitative phase imaging. Principal component analysis have showed clear differentiation of samples in order to classify the control erythrocytes and the tonicity stressed erythrocytes. Present study has also demonstrated the application of Raman Tweezers spectroscopy as a potential tool for probing red blood cell under different stress conditions.