We developed a unique analytical technique for the evaluation of Entecavir monohydrate (ETV) in its pharmaceutical dosage form using derivative spectroscopy assisted FTIR. This approach requires the formation of solid pellets of Entecavir using potassium bromide (KBr) with the aid of geometrical mixing. The spectra were calculated by direct measurement technique using reduced path length in the absorbance mode, and the equipment was configured to secure it at 8cm-1 resolution. We scanned the spectra between the ranges of 4000 to 400 cm-1. FTIR spectra drug exhibited overlapped functional group peaks with baseline correction at 1631 cm-1 corresponding to C=O stretching. From these FTIR spectra, we detected intense, clear, and proportional second derivative peaks between 1639.38 and 1620.09 cm-1. These peaks, in the range of concentration 12.5-200 μg/mg, obeyed Beer-Lambert’s law. Therefore, we elected C=O stretching for the quantitative evaluation of Entecavir employing second-order derivative spectroscopy.