- A photon diffusion model has been developed to calculate the steady-state spatially resolved fluorescence from pencil beam excitation in layered tissue. The model allows the calculation of both the excitation reflectance and the fluorescence escape for an arbitrary continuous depth distribution of tissue optical properties and fluorophore concentration. The validity of this model was verified by comparison with Monte Carlo simulations and experimental measurements using phantoms with tissue-like optical properties. The potential usefulness of the spatially resolved fluorescence was explored using the model and simulations of realistic drug distributions. It was shown that using this technique it may be possible to quantify the diffusion of a topically administered drug into the skin, or the photobleaching of a sensitizer during photodynamic therapy.