Determination of tissue optical properties is fundamental for application of light in either therapeutical or diagnostics procedures. In the present work we implemented a spatially resolved steady-state diffuse reflectance method where only two fibers (one source and one detector) spaced 2.5 mm apart are used for the determination of the optical properties. The method relies on the spectral characteristics of the tissue chromophores (water, dry tissue, and blood) and the assumption of a simple wavelength dependent expression for the determination of the reduced scattering coefficient. Because of the probe dimensions the method is suited for endoscopic measurements. The method was validated against more traditional models, such as the diffusion theory combined with adding doubling for in vitro measurements of bovine muscle. Mean and standard deviation of the absorption coefficient and the reduced scattering coefficient at 630 nm for normal mucosa were 0.87±0.22 cm−1 and 7.8±2.3 cm−1, respectively. Cancerous mucosa had values 1.87±1.10 cm−1 and 8.4±2.3 cm−1, respectively. These values are similar to data presented by other authors. Blood perfusion was the main variable accounting for differences in the absorption coefficient between the studied tissues. © 2005 Society of Photo-Optical Instrumentation Engineers.