The optical properties of cationic dicarbocyanide dyes have been shown to depend on the transmembrane electrical potential of liposomes or living cells. In this work, we developed the most complete computational model allowing optimization of experimental conditions to monitor the membrane potential of membrane vesicles composed of neutral and charged phospholipids at fixed ionic strength of incubation media in a desirable voltage range. The model allows predicting potential‐dependent changes of the fluorescent properties of DiSC3(5) on concentration of the dye and on the lipid composition of the liposomes of certain sizes. It also takes into account the values of the surface potentials of the inner and outer leaflets of the lipid bilayer, as well as their changes due to the DiSC3(5) incorporation into the lipid phase. The probe dimerization in the aqueous and lipid phases, and the possibilities of the voltage‐dependent transitions of the dimers between two leaflets of the lipid bilayer and between the aqueous and lipid phases were also included in the model, in addition to the monomer transitions. The model was adjusted to several literature data obtained with DiSC3(5) probe as an indicator of the membrane potential in experiments with liposomes and biomembrane vesicles. Colciencias (Colombia) research grants #111840820380 and #111852128625.