The thymidine analogue 3'-deoxy-3'-[18F]fluorothymidine, or [18F]fluorothymidine ([18F]FLT), is used to measure tumor cell proliferation with positron emission tomography (PET) imaging technology in nuclear medicine. FLT is phosphorylated by thymidine kinase 1 (TK1) and then trapped inside cells; it is not incorporated into DNA. Imaging with 18F-radiolabeled FLT is a noninvasive technique to visualize cellular proliferation in tumors. However, it is difficult to distinguish between [18F]FLT and its metabolites by PET imaging, and quantification has not been attempted using current imaging methods. In this study, we successfully acquired in vivo19F spectra of natural or nonradioactive 3'-deoxy-3'-fluorothymidine ([19F]FLT) and its monophosphate metabolite (FLT-MP) in a tumor xenograft mouse model using 9.4T magnetic resonance imaging (MRI). This preliminary result demonstrates that 19F magnetic resonance spectroscopy (MRS) with FLT is suitable for the in vivo assessment of tumor aggressiveness and for early prediction of treatment response.