Abnormally shaped red blood cells (RBCs), called poikilocytes, can cause anemia. At present, the biochemical abnormalities in poikilocytes are not well understood. Normal RBCs and poikilocytes were analyzed using whole-blood and single-cell methods. Poikilocytes were induced in rat blood by intragastrically administering titanium dioxide (TiO₂) nanoparticles. Complete blood count and inductively coupled plasma mass spectrometry analyses were performed on whole-blood to measure average RBC morphology, blood hemoglobin (HGB), iron content, and other blood parameters. Follow-up confocal Raman spectroscopy was performed on single RBCs to analyze cell-type-specific HGB content. Two types of poikilocytes, acanthocytes and echinocytes, were observed in TiO₂ blood samples, along with normal RBCs. Acanthocytes (diameter 7.7  ±  0.5  μm) and echinocytes (7.6  ±  0.6  μm) were microscopically larger (p  <  0.05) than normal RBCs (6.6  ±  0.4  μm) found in control blood samples (no TiO₂ administration). Similarly, mean corpuscular volume was higher (p  <  0.05) in TiO₂ whole-blood (70.70  ±  1.97  fl) than in control whole-blood (67.42  ±  2.03  fl). Poikilocytes also had higher HGB content. Mean corpuscular hemoglobin was higher (p  <  0.05) in TiO₂ whole-blood (21.84  ±  0.75  pg) than in control whole-blood (20.8  ±  0.32  pg). Iron content was higher (p  <  0.001) in TiO₂ whole-blood (697.0  ±  24.5  mg  /  l) than in control whole-blood (503.4  ±  38.5  mg  /  l), which supports elevated HGB as iron is found in HGB. HGB-associated Raman bands at 1637, 1585, and 1372  cm^−  1 had higher (p  <  0.001) amplitudes in acanthocytes and echinocytes than in RBCs from control blood and normal RBCs from TiO₂ blood. Further, the 1585-cm^−  1 band had a lower (p  <  0.05) amplitude in normal RBCs from TiO₂ versus control RBCs. This represents biochemical abnormalities in normal appearing RBCs. Overall, poikilocytes, especially acanthocytes, have elevated HGB.