A sensitive spectrofluorimetric method was developed for the determination of tizanidine in human plasma, urine and pharmaceutical preparations. The method is based on reaction of tizanidine with 1-dimethylaminonaphthalene-5-sulphonyl chloride (dansyl chloride) in an alkaline medium to form a highly fluorescent derivative that was measured at 511?nm after excitation at 383?nm. The different experimental parameters affecting the fluorescence intensity of tizanidine was carefully studied and optimized. The fluorescenceconcentration plots were rectilinear over the ranges 50500 and 20300?ng/mL for plasma and urine, respectively, detection limits of 1.81 and 0.54?ng/mL and quantification limits of 5.43 and 1.62?ng/mL for plasma and urine, respectively. The method presents good performance in terms of linearity, detection and quantification limits, precision, accuracy and specificity. The proposed method was successfully applied for the determination of tizanidine in pharmaceutical preparations. The results obtained were compared with a reference method, using t- and F-tests. Copyright (c) 2011 John Wiley & Sons, Ltd.