The recent epidemic of Zika virus (ZIKV) in the Americas and its association with fetal and neurological complications has shown the need to develop a treatment. Repurposing of drugs that are already FDA approved or in clinical development may shorten drug development timelines in case of emerging viral diseases like ZIKV. Initial studies have shown conflicting results when testing sofosbuvir developed for treatment of infections with another Flaviviridae virus, hepatitis C virus. We hypothesized that the conflicting results could be explained by differences in intracellular processing of the compound. We assessed the antiviral activity of sofosbuvir and mericitabine against ZIKV using Vero, A549, and Huh7 cells and measured the level of the active sofosbuvir metabolite by mass spectrometry. Mericitabine did not show activity, while sofosbuvir inhibited ZIKV with an IC50 of ˜4 μM, but only in Huh7 cells. This correlated with differences in intracellular concentration of the active triphosphate metabolite of sofosbuvir, GS-461203 or 007-TP, which was 11–342 times higher in Huh7 cells compared to Vero and A549 cells. These results show that a careful selection of cell system for repurposing trials of prodrugs is needed for evaluation of antiviral activity. Furthermore, the intracellular levels of 007-TP in tissues and cell types that support ZIKV replication in vivo should be determined to further investigate the potential of sofosbuvir as anti-ZIKV compound.