In this study, a new, fast and sensitive HPLC method with fluorometric detection was developed for the determination of mesalazine in human plasma and applied to a pharmacokinetic study. Mesalazine was precolumn derivatized with NBD-Cl and the fluorescent derivative was separated on a C18 (150 x 4.6 mm x 2.6 mu m) analytical column at 30 oC using a mobile phase composed of acetonitrile-0.1% o-phosphoric acid in water (70:30, v/v) by isocratic elution with flow rate of 1.0 mL min(-1). The method was based on the measurement of the derivative using fluorescence detection (lambda(ex) = 280 nm, lambda(em) = 325 nm). The retention time of mesalazine is 3.08 +/- 0.06 min. Nortriptiline was used as internal standard. This currently developed method was validated according to ICH criteria by evaluating the specificity, linearity, precision, accuracy and robustness. The method was determined to be linear in a concentration range of 0.25-1.5 mu g mL(-1) with the correlation coefficient of 0.9997. LOD and LOQ were found to be 0.075 and 0.25 mu g mL(-1), respectively. Intraday and interday RSD values were less than 5.92%. The plasma concentration-time profile and pharmacokinetic parameters such as AUC(0-t), AUC(0-infinity), C-max, t(max), t(1/2), were calculated according to the assays. The presented method can certainly be used for bioequivalence and bioavailability investigations and routine analysis of the drug in plasma.