In microdose clinical trials a maximum of 100 μg of drug substance is administered to participants, in order to determine the pharmacokinetic properties of the agents. Measuring low plasma concentrations after administration of a microdose is challenging and requires the use of ulta-sensitive equipment. Novel liquid chromatography-mass spectrometry (LC–MS/MS) platforms can be used for quantification of low drug plasma levels. Here we describe the development and validation of an LC–MS/MS method for quantification of gemcitabine and its metabolite 2′,2′-difluorodeoxyuridine (dFdU) in the low picogram per milliliter range to support a microdose trial. The validated assay ranges from 2.5–500 pg/mL for gemcitabine and 250–50,000 pg/mL for dFdU were linear, with a correlation coefficient (r2) of 0.996 or better. Sample preparation with solid phase extraction provided a good and reproducible recovery. All results were within the acceptance criteria of the latest US FDA guidance and EMA guidelines. In addition, the method was successfully applied to measure plasma concentrations of gemcitabine in a patient after administration of a microdose of gemcitabine.