We evaluated usability of a previously developed genetically encoded molecular crowding sensor in various biological phenomena. Molecular crowding refers to intracellular regions that are occupied more by proteins and nucleotides than by water molecules and is thought to have a strong effect on protein function. To evaluate intracellular molecular crowding, usually the diffusion coefficient of a probe is used because it is related to mobility of the surrounding molecular crowding agents. Recently, genetically encoded molecular crowding sensors based on Förster resonance energy transfer were reported. In the present study, to evaluate the usability of a genetically encoded molecular crowding sensor, molecular crowding was monitored during several biological events. Changes in molecular crowding during stem cell differentiation, cell division, and focal adhesion development and difference in molecular crowding in filopodia locations were examined. The results show usefulness of the genetically encoded molecular crowding sensor for understanding the biological phenomena relating to molecular crowding.