Synchrotron radiation-based Fourier transform infrared spectromicroscopy is a newly emerging analytical tool capable of monitoring the biochemistry within an individual living mammalian cell in real time. This unique technique provides infrared (IR) spectra, hence chemical information, with high signal to noise at spatial resolutions as fine as 3–10 µm. Mid-IR photons are too low in energy (0.05–0.5 eV) to either break bonds or to cause ionization, and the synchrotron IR beam has been shown to produce minimal sample heating. However, an important question remains, ‘‘Does the intense synchrotron beam induce any cytotoxic effects in living cells?’’ In this work, we present the results from a series of standard biological assays to evaluate any short- and/or long-term effects on cells exposed to the synchrotron radiation-based infrared (SR-IR) beam. Cell viability was tested using alcian blue dye exclusion and colony formation assays. Cell-cycle progression was tested with bromodeoxyuridine (BrdU) uptake during DNA synthesis. Cell metabolism was tested using a 3-[4,5- Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. All control, 5, 10, and 20 min SR-IR exposure tests (267 total and over 1000 controls) show no evidence of cytotoxic effects. Concurrent infrared spectra obtained with each experiment confirm no detectable biochemical changes between control and exposed cells.