AbstractThe photon counting histogram (PCH) analysis is a fluorescence fluctuation method that is able to characterize the brightness and concentration of different fluorescent species present in a liquid sample. We find that the PCH model using a three‐dimensional Gaussian observation volume profile is inadequate for fitting experimental data obtained from a confocal setup with one‐photon excitation. We propose an improved model, which is based on the correction to the observation volume profile for the out‐of‐focus emission. We demonstrate that this model is able to resolve different species present under a wide range of conditions. Attention is given to how this model allows the examination of the effects of different instrumental setups on the resolvability.