Gemcitabine is an established chemotherapy agent in several solid tumors. Its mechanism of action has been theoretically established and this is supported with strong experimental evidence. However, certain aspects of the resistance mechanism for this agent remain elusive. We present a method of analysis using tandem liquid chromatography and mass spectrometry that provides a broader, yet more focused view of the action of gemcitabine and its primary metabolite, difluorodeoxyuridine in relation to the (deoxy) nucleoside and (deoxy) nucleotide pools in tumor cell lines. Alcoholic cytosole extracts were incubated with alkaline phosphatase reducing the nucleotide pools to their respective nucleosides. Determination of the nucleoside content by a sensitive LCMSMS method before and after incubation enables the calculation of the total amount of phosphorylation of each (deoxy) nucleoside in the cell. Incubation with clinically relevant levels of gemcitabine (dFdC) or difluorodeoxyuridine (dFdU) for 24 hours enabled the determination of the changes in the (deoxy) nucleotide pools in relation to chemotherapeutic and toxicological effects. Confirmation of the presence of dFdC phosphorylation is presented as well as direct evidence of dFdU phosphorylation after both dFdC and dFdU treatment. Differences in the nucleotide pools are presented after dFdC and dFdU incubation, indicating that dFdU might have more chemotherapeutic properties than previously believed.