Fluorescence microscopy does constitute an essential tool in cellular and molecular biology research as well as in materials science. This technique permits to study both living cells dynamics in a noninvasive way or fixed cells, providing large amount of quantitative information. However, the technique is significantly affected by the photobleaching of fluorescent substances. This phenomenon is a photochemical transformation that occurs in different extent, in fluoresce molecules, and diminishing its ability to produce fluorescence, and thus reducing the signal to noise ratio in final images. In order to establish a model that allows explaining the phenomenon and providing information for the quantitative analysis of images, we analyzed four commonly models used in microscopy of exponential fluorescence decay. These were compared objectively using analysis of variance, being the function of four-parameter bi-exponential without constant term the model that best fit the data.