For many clinical light applications, such as photodynamic therapy (PDT), the therapeutic effect strongly depends on the light dose in a certain tissue depth. A measure for the attenuation and penetration of light in tissue is the optical penetration depth, which is derived from the tissue’s optical properties at a certain wavelength. Therefore, in vivo measurements to determine the optical properties were performed of the bladder wall (n = 12) and brain tissue (n = 11) on patients undergoing photodynamic therapy. The tip of a 400 μm bare fiber was placed in contact with the investigated tissue, either during open surgery (brain) or through the working channel of a cystoscope (bladder wall). Light of the wavelengths 420-450 nm, 532 nm, and 635 nm was coupled alternately into the fiber. The diffuse backscattered light was detected spatially resolved by means of a CCD camera. Additionally, the total diffuse reflectance of the tissue site was determined, by relating the white light spectra remitted from the tissue to that of a reflectance standard. These two independent measurements were fitted with Monte Carlo simulations. Thus, the reduced scattering and absorption coefficient could be obtained and the optical penetration depth was derived. The presented investigations showed that spatially resolved diffuse reflectance in combination with total diffuse remission provides a valuable method to determine tissue optical properties in vivo. Two human organs were analyzed with this technique and both, bladder wall tissue and brain tissue showed reproducible results.