A practical high-performance liquid chromatographic method with fluorometric detection for the determination of guaifenesin and dextromethorphan in cough syrup is described. The developed method contains simple, accurate and rapid sample preparation steps for the separation and simultaneous determination of active compounds in samples with existence of other excipients. Amilorid (AM) was used as an internal standard (IS) that has a fluorescence character in the working wavelength region. A good chromatographic separation of two drugs and IS was achieved on a Waters Symmetry(R) C18 Column 5 mu m 4.6 x 250 mm with mobile phase consisting of phosphate buffer (0.2 M, pH=2), acetonitrile and methyl alcohol (v/v, 62:23:15). Eluents were monitored by fluorescence detection at the excitation and emission wavelengths of 277 and 588 nm, respectively. Under the described conditions, the obtained calibration graphs are linear over the concentration range of 0.054.2 and 1.2-2.4 mg/mL for dextromethorphan and guaifenesin, respectively and the regression coefficients were greater than 0.999. The validation study was carried out fulfilling the ICH guidelines in order to prove that the new analytical method meets the fundamental criteria including selectivity, linearity, precision, accuracy and sensitivity.