In this work, we employed the Raman microscopy to study the internalization kinetics and spatial distribution of small interfering RNA (siRNA)-diatomite nanoparticles (DNPs) complex in human lung epidermoid carcinoma cell line (H1355) up to 72 h. Raman images are compared with confocal fluorescence microscopy results. The Raman analysis provides that the siRNA-DNPs are internalized and co-localized in lipid vesicles within 18 h, after that equilibrium is achieved.