A new and sensitive method was developed for the determination of atomoxetine in human plasma and urine using a reversed phase separation followed by pre-column derivatization with 1-dimethylaminonaphthalene-5-sulphonyl chloride (dansyl chloride) and fluorescence detection. The liquid chromatographic separation was achieved on a Inertsil C-18 column using methanol:water (85:15 v/v) as a mobile phase in an isocratic elution mode. The eluents were monitored by a fluorescence detector set at 375 and 537 nm of excitation and emission wavelengths, respectively. Mexiletine was used as an internal standard. The method was validated for linearity, limit of detection, limit of quantification, precision, accuracy, recovery, robustness, and system suitability. The method was linear over the concentration range of 10-2250 and 25-3000 ng mL(-1) for plasma and urine, respectively. The mean recovery of atomoxetine from plasma and urine was 97.39 and 96.77%, respectively.