A method for estimating the optical properties of two-layered media (such as squamous epithelial tissue) over a range of wavelengths in the ultraviolet–visible spectrum is proposed and tested with Monte Carlo modeling. The method first used a fiber-optic probe with angled illumination and the collection fibers placed at a small separation \left(\le 300\text{\hspace{0.17em} μm}\right) to restrict the transport of detected light to the top layer. A Monte Carlo-based inverse model for a homogeneous medium was employed to estimate the top layer optical properties from the measured diffuse reflectance spectrum. Then a flat-tip probe with a large source-detector separation \left(\ge 1000\text{\hspace{0.17em} μm}\right) was used to detect diffuse reflectance preferentially from the bottom layer. A second Monte Carlo-based inverse model for a two-layered medium was applied to estimate the bottom layer optical properties, as well as the top layer thickness, given that the top layer optical properties have been estimated. The results of Monte Carlo validation show that this method works well for an epithelial tissue model with a top layer thickness ranging from 200\text{\hspace{0.17em} to \hspace{0.17em}}500\text{\hspace{0.17em} μm} . For most thicknesses within this range, the absorption coefficients were estimated to within 15\% of the true values, the reduced scattering coefficients were estimated to within 20\% and the top layer thicknesses were estimated to within 20\% . The application of a variance reduction technique to the Monte Carlo modeling proved to be effective in improving the accuracy with which the optical properties are estimated.