A simple, rapid, cost-effective and green high-performance liquid chromatographic assay for determination of Gemcitabine in human plasma using a C18 reversed-phase analytical column was developed and validated. The separation was conducted by means of a mobile phase composed of formic acid solution (pH = 3): ethanol (55:45) running at a flow-rate of 1.0 mL min-1 with UV detection at 310 nm. The column temperature was set at 50 °C. Sample preparation involved protein precipitation by zinc sulfate-ethanol solution. This method is consistent with a high recovery of Gemcitabine in human plasma ranging from 95.98 to102.50 %.The calibration curves were linear over concentration range of 0.05-10.00 mL-1(r2> 0.9999). Between- and within-day variability was less than 15 % and the bias was within ±15 %. This validated method was successfully applied to a pharmacokinetic study enrolling seven cancer patients after administration of a morning oral dose of 1500 mg.