A high-performance liquid chromatographic method with fluorescence detection (HPLC–FLD) for the determination of pyrene in rat plasma was developed and validated. The method used fluorene as internal standard (IS), following a single-step protein precipitation, the analyte and internal standard were separated on a C18 column with a mobile phase containing methanol–water (90:10, v/v) at a flow rate of 1 ml/min. The analytes were detected by using fluorescence detection at an excitation and emission wavelength of 265 and 394 nm, respectively. Two calibration curves were constructed in the range of 2–100 ng/ml and 0.1–5 μg/ml for pyrene with a lower limit of quantitation (LLOQ) of 2 ng/ml. Both intra-day and inter-day precision were less than 6% except at LLOQ, for which the precision was 10.6 and 9.8, respectively. Accuracy ranged from 98.3 to 103.6%, except at LLOQ, for which the accuracy was about 85%. The recovery ranged from 84.7 to 95.0% at the low, medium and high concentrations. The present HPLC–FLD method was rapid, sensitive, and reliable. The method described herein had been successfully applied for the pharmacokinetic studies in female Wistar rats after administration of 10 mg equivalent pyrene/kg dose of solution of pyrene and 1 mg equivalent pyrene/kg dose of pyrene-loaded nanoparticle.