In this study, a ratiometric quantification method is developed and applied to monitor mesotetra(hydroxyphenyl) chlorin (mTHPC) pharmacokinetics in the rat skin-fold observation chamber. The method employs a combination of dual-wavelength excitation and dual-wavelength detection. The excitation and detection wavelengths were selected in close to NIR. The first excitation wavelength was used to excite the mTHPC and autofluorescence and the second to excite only autofluorescence, so that this could be substracted. Subsequently the difference was divided by the autofluorescence. Since the method applies division of signal with no mTHPC fluorescence, theory suggests on linear dependency of the method on photosensitizer concentration.