Nuclear magnetic resonance spectroscopy of fluorine-19 ((19)F NMR) has proven useful for evaluating kinetics of fluorinated chemotherapy drugs in tumors in vivo. This work investigated how three perfusion-enhancing vascular modifiers (BQ123, thalidomide, and Botulinum neurotoxin type A [BoNT-A]) would affect the chemotherapeutic efficacy of gemcitabine, a fluorinated drug widely used in human cancer treatment. Murine tumor growth experiments demonstrated that only BoNT-A showed a strong trend to enhance tumor growth inhibition by gemcitabine (1.7 days growth delay, P = 0.052, Student t-test). In accord with these results, (19)F NMR experiments showed that only BoNT-A increased significantly the uptake of gemcitabine in tumors (50% increase, P = 0.0008, Student t-test). Further experiments on gemcitabine kinetics (NMR vs time) and distribution ((19)F MRI) confirmed the uptake-enhancing properties of BoNT-A. The results of this study demonstrate that (19)F NMR can monitor modulation of the pharmacokinetics of fluorinated chemotherapy drugs in tumors. The results also show that (19)F NMR data can give a strong indication of the effectiveness of perfusion-enhancing vascular modifiers for improving gemcitabine chemotherapy in murine tumors. (19)F NMR is a promising tool for preclinical evaluation of such vascular modifiers and may ultimately be used in the clinic to monitor how these modifiers affect chemotherapy.