The use of spectrophotometer in analytical chemistry of mixtures is limited by overlap of absorbance spectrum of the components of the mixture. The absorbance of a mixture at a particular wavelength is the sum–total of the absorbance of all the analytes in the mixture at the same wavelength. Determination of concentration of one analyte in the mixture will be overestimated due to absorbance of other analytes (herein known as interferents). The estimation method can be imprecise (due to varying content of interferents) and always inaccurate (due to presence of interferents). In such spectrophotometric method involving multiple analytes, derivative spectroscopy can be used to ameliorate failed accuracy validation. It uses first or higher derivative of absorbance with respect to wavelength for both qualitative and quantitative discrimination. The derivative bands are sharper and separate the overlapping bands. The peaks of derivative spectra can be used to construct the calibration curves for determination of the concentration of the analyte of interest in the mixture. In this article, we describe a computer simulated model of derivative spectroscopy for estimation of concentration of the individual drugs in a solution.