Authors developed the computer application program (language: APPLE SOFT BASIC) for derivative spectrophotometry. By means of this program, derivative of spectral absorbance with respect to wavelength is recorded versus wavelength. To try this program in connection with spectrophotometer system, the authors have done the simultaneous quantitation of pyridoxine center dot HCl and nicotinamide in the mixture, and the result was compared with that of absorbance method.