Gemcitabine is an anticancer agent acting against several solid tumors. It requires nucleoside transporters for cellular uptake and deoxycytidine kinase for activation into active gemcitabine-triphosphate, which is incorporated into the DNA and RNA. However, it can also be deaminated in the plasma. The intracellular level of gemcitabine-triphosphate is affected by scheduling or by combination with other chemotherapeutic regimens. Moreover, higher concentrations of gemcitabine-triphosphate may affect the toxicity, and possibly the clinical efficacy. As a consequence, different nucleoside analogs have been synthetized with the aim to increase the concentration of gemcitabine-triphosphate into cells. In this review, we summarize currently published evidence on pharmacological factors affecting the intracellular level of gemcitabine-triphosphate to guide future trials on the use of new nucleoside analogs.