A comparison of two spectrophotometric methods and a HPLC method were described in this work for the analysis of pyridoxine hydrochloride and thiamine hydrochloride in a vitamin combination. In the first method, A1/1 (1%, 1 cm) values of these two compounds were calculated using absorbances measured at 246.8 and 290.5 nm in zero-order spectra. The matrix was written for A1/1 (1%, 1 cm) values and the concentration of both compounds were determined using 'Matlab' software. In the second method, the measurements in the derivative of the ratio spectra were made at 297.8 and 309.5 nm for pyridoxine hydrochloride and at 245.6 and 257.7 nm for thiamine hydrochloride. The calibration graphs were established in the range 8-40 μg/ml of both vitamins. In the HPLC method, the separation of these compounds was realized on a Nucleosil 100-5 C18 column with 0.1 M (NH4)2CO3-water-methanol (5:15:80 v/v) as the mobile phase. Results of spectrophotometric and HPLC procedures were compared. (C) 2000 Elsevier Science B.V.