Abstract: Fluorescent probes are widely used in membrane biophysical studies to investigate the properties of the molecular environment of cell membrane and biochemical processes related to it. These probes provide information of the molecular environment trough changes of their photophysical properties. While these probes can provide valuable information of the membrane molecular properties, a clear structural description, at the molecular scale, of the membrane properties surrounding the fluorescent molecular probe, is essential for the interpretation of the experimental fluorescent signal. In this work, we studied the membrane permeation properties of four benzo[a]phenoxazinium fluorescent probes with different alkyl chains in the tetracyclic ring in DPPC membranes using molecular modelling techniques . We describe the molecular mechanism of interaction between the molecular probes and the DPPC lipids. We show that probes can be preferentially located at the membrane water interface interaction with the lipid polar groups, while others can be cross the membrane in a passive way. This processes is dependent on the tetracyclic substitutions and alkyl chain present in the fluorescent probe. These findings can provide a detailed rational of the structural properties surrounding the molecular probes that could be correlated with the experimentally measured fluorescence.