Abstract We present a comprehensive and analytical treatment of continuous photobleaching in a compartment, under single photon excitation. In the very short time regime ( t < 0.1 ms), the diffusion does not play any role. After a transition (or short time regime), one enters in the long time regime ( t > 0.1–5 s), for which the diffusion and the photobleaching balance each other. In this long time regime, the diffusion is either fast (i.e., the photobleaching probability of a molecule diffusing through the laser beam is low) so that the photobleaching rate is independent of the diffusion constant and dependent only of the laser power, or the diffusion is slow (i.e., the photobleaching probability is high) and the photobleaching rate is mainly dependent on the diffusion constant. We illustrate our theory by using giant unilamellar vesicles ranging from ∼10 to 100 μm in diameter, loaded with molecules of various diffusion constants (from 20 to 300 μm 2/s) and various photobleaching cross sections, illuminated under laser powers between 3 and 100 μW. We also demonstrated that information about compartmentation can be obtained by this method in living cells expressing enhanced green fluorescent proteins or that were loaded with small FITC-dextrans. Our quantitative approach shows that molecules freely diffusing in a cellular compartment do experience a continuous photobleaching. We provide a generic theoretical framework that should be taken into account when studying, under confocal microscopy, molecular interactions, permeability, etc.