In this study, a simple, efficient and rapid Ultra High Performance Liquid Chromatography method with fluorescence detection (UHPLC-FLD) has been developed and validated for the determination of Ochratoxin-A (OTA) in rat brain microdialysates and plasma samples. Six adult male wistar rats were used in the study and a single dose (5 mg/kg b.w.) of OTA was given by intraperitoneal (i.p.) injection. Rat blood and microdialysate samples were collected simultaneously after i.p. injection in awake freely moving rats, over a twelve-hour period. An UHPLC analysis was performed on a Zorbax Eclipse Plus C8 (150 mm x 3.0 mm ID x 1.8 mu m particles) column with a mobile phase of acetonitrile:water:phosphoric acid (50:50:0.1, v/v) using a flow rate of 0.6 mL/min. The fluorescence detector was set at 330 nm excitation and 460 nm emission wavelengths. Diflunisal (DIF) was used as an internal standard (IS). OTA and IS were separated within 5 min under these conditions. The method was validated in terms of linearity, precision, accuracy, limit of detection, limit of quantification, and stability. Calibration curves obtained with spiked biological matrices show good linearity with high correlation coefficients. The intra- and inter-day assay variability was < 5% for the OTA. The limit of detection and the limit of quantification values were found to be 0.490 ng/mL and 1.48 ng/mL for plasma; 0.0900 ng/mL and 0.270 ng/mL for microdialysate samples, respectively. This method was successfully applied for the monitoring of OTA levels in the rat brain and plasma samples.