Frequency response analysis via pulse testing for engineering systems and near infrared (NIR) time resolved spectroscopy (TRS) for biological system characterization involve identical principles: the system of interest is disturbed by an input pulse and the output response is observed. Since a sharp pulse, such as the Dirac delta function, contains the information of multimodulation frequencies (theoretically from 0 to infinity in frequency) a narrow pulse TRS input can produce a wide range frequency response for identifying any system of interest. Currently used NIR-TRS spectral analyses either fit the spectra with a known theoretical solution or use photon mean time-of-flight. Transforming the system time domain representation to the frequency domain generates five system parameters that can be valuable for process identification utility: steady state gain, time constant, system order, and magnitude ratio and phase shift over a wide frequency range. Optical contrast agents or fluorescent agents can be used to enhance the capability of optical instruments in detecting biological heterogeneities. In this article, magnitude ratio, phase shift, and other system parameters derived from the transfer function of systems with both a fluorescent absorber and a regular absorber are correlated with the position of the absorber. This technique is important in that ultimately it can be used to enhance optical medical imaging.