The microsphere formation mechanism of bis(N alpha-amido-L-phenylalanine) 1,1-cyclobutanedicarboxylate, whose self-assembly was triggered by protonation of its carboxylic groups, was investigated by Raman microscopy. An image of the Raman intensity contour map, Raman imaging, shows that water is excluded from the core during the L-phenylalanine dicarboxylate aggregation. In situ Raman microscopic studies on the size and morphological changes of microspheres indicates that osmotic pressure on the sphere, generated by water exclusion from the sphere＇s core, controls the final assembled structure and morphology.