The Raman Deuterium Isotope Probing (Raman-DIP) method was applied to study the response of human breast adenocarcinoma cells (the MCF-7 cells) to the GSK2334470 drug in vitro. The cytotoxicity level of the GSK2334470 to the MCF-7 cell evaluated by the Raman-DIP method and CCK-8 assay was highly matched, as both methods suggested that 10 μM was the cytotoxic concentration for inhibition of cell regeneration. In additional, the relationship between drug dose and the cell response (RSI) were fitted, showing that our experimental data were consistent with the dose-response relationship, and the Raman-DIP could be applied for sensitivity testing of targeted anti-cancer drugs. The variation of the bio-macromolecules reflected by the Raman signal in the fingerprint range was closely related to the GSK2334470 concentration. The Raman-DIP method can monitor the inhibition of the GSK2334470 to the MCF-7 cell proliferation with high accuracy as the CCK-8 assay. The combination of Raman-DIP results and fingerprint range information can effectively improve the evaluation, serving as a supplement for the existing anti-cancer drug sensibility assay. This method is valuable to reveal the intracellular macromolecules changes related to the drug treatment. K E Y W O R D Santi-cancer drug screening, breast cancer, deuterium isotope probing, Raman spectroscopy, single cell level Xia Huang and Chengjian Li contributed equally to this work and should be considered co-first authors.