Extraction of quantitative biochemical information from measured fluorescence spectra is hindered by the presence of potentially significant distortions introduced by tissue scattering and absorption. Such distortions can be removed by extracting the intrinsic fluorescence spectra from the measured fluorescence spectra. This paper explores the potential applicability of spatially resolved fluorescence technique for simultaneous extraction of intrinsic fluorescence and evaluation of optical transport parameters, namely, reduced scattering coefficient (μs|), absorption coefficient (μa) from tissue mimicking model systems and human breast tissues. A hybrid diffusion theory-Monte Carlo simulation based theoretical model was used to estimate the values for μs| and μa and to recover intrinsic fluorescence from the measured spatially resolved fluorescence from the samples. The agreement between the values for μs' and μa estimated for tissue mimicking phantoms using the spatially resolved fluorescence measurement technique and the corresponding calculated values are seen to be satisfactory with a maximum percentage error of ≤ 10 % and also line shape and intensity of intrinsic fluorescence recovered using this approach was observed to be free from the disentangling effects of absorption and scattering properties of the medium. Intrinsic fluorescence spectra of breast tissues show a distinct difference between malignant and its normal counterpart. A narrowing of the line shape is also observed as compared to the bulk fluorescence spectrum.© (2006) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.