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Herein, Raman and surface-enhanced Raman spectroscopies (SERS) were successfully employed to establish the chemical interactions of citrate-capped silver nanoparticles (AgNPs, 10–15 nm) with human red blood cells (RBCs). The Raman/SERS spectra offered spectral evidence for the cellular uptake of AgNPs and the subsequent change in the conformation of the most abundant component, hemoglobin (Hb), from oxyhemoglobin to deoxyhemoglobin. The spectral characterization of AgNPs' interactions with other RBC biomarkers (membrane proteins and lipids) was impeded by the dominant Hb bands, even for non-resonant Hb conditions. CytoViva hyperspectral imaging and graphite furnace atomic absorption spectroscopy (GFAAS) served as complementary tools to effectively address the challenges related to a single excitation line (632.8 nm) and the resolution of the confocal Raman microscope (0.5–1.0 µm). CytoViva confirmed the RBC-AgNP interactions through hyperspectral signatures and facilitated the label-free localization of AgNPs extracellularly and intracellularly. Irreversible agglutination of RBCs was noted after 24 h of exposure, raising concerns about the toxicity of AgNPs of biocompatible citrate coatings. GFAAS validated the Raman/SERS results by quantifying the proportion of AgNPs absorbed by RBCs, which was significant (~48% AgNPs by mass), mostly at the membrane (60% RBCs), and size dependent (no large AgNPs or AgNP-aggregates in RBCs, after 12–24 h). [ABSTRACT FROM AUTHOR]