The feasibility of using quantitative diffuse reflectance spectroscopy to longitudinally monitor physiological response to cancer therapy was evaluated in a preclinical model. This study included two groups of nude mice bearing 4T1 flank tumors (N = 50), half of which were treated with a maximum tolerated dose of doxorubicin (DOX). Diffuse reflectance spectra were collected from tumors during a period of 2 weeks using a fiber-optic probe coupled to a spectrometer. These spectra were quantified using an inverse scalable Monte Carlo model of light transport in tissue to extract the concentrations of oxygenated, deoxygenated hemoglobin (dHb), and a wavelength mean reduced scattering coefficient (<micro(s)'>). The tumor growth rates of the treated and control groups were nearly identical, as were changes in the scattering parameter <micro(s)'> during this time frame. However, tumors treated with DOX showed a transient but significant increase in blood oxygen saturation. A comparison between the optically derived and immunohistochemical end points in a subset of the 50 animals showed that the temporal kinetics of dHb concentration and <micro(s)'> were highly concordant with those of hypoxic and necrotic fractions, respectively. In conclusion, optical methods could function as a "screening" technology in longitudinal studies of small animal tumor models to accelerate development and testing of new anticancer drugs. This technique could isolate specific landmark time points at which more expensive and sophisticated imaging methods or immunohistochemistry could be performed.