We report the first Raman spectra of individual living and dead cells (MLE‐12 line) cultured on bioinert standard poly‐L‐lysine coated fused silica and on bioactive 45S5 Bioglass^® measured at 785 nm laser excitation. At this excitation wavelength no damage was induced to the cells even after 40 minutes irradiation at 115 mW power, as indicated by cell morphology observation and trypan blue viability test. We show that shorter wavelength lasers, 488 nm and 514 nm, cannot be used because they induce damage to the cells at very low laser powers (5 mW) and short irradiation times (5–20 minutes). The most important differences between the spectra of living and dead cells are in the 1530–1700 cm⁻¹ range, where the dead cells have strong peaks at 1578 cm⁻¹ and 1607 cm⁻¹. Other differences occur around the DNA peak at 1094 cm⁻¹. Our study establishes the feasibility of using the 785 nm laser for an in situ real‐time non‐invasive method to follow biological events (proliferation, differentiation, cell death, etc.) within individual cells cultured on bioactive scaffolds in their physiologic environment over long periods of time.