In this paper, we present an in situ visualization approach for investigating the formation mechanism of the imprinting process for patterning microstructures on a glass substrate. In the experimental study, K-PG375 was used as a glass substrate, and a mold with V-groove patterns was fabricated on glassy carbon. The microstructure profile corresponding to relative process parameters during imprinting was visualized using the proposed measurement system. Meanwhile, the finite element method (FEM) was also utilized to predict the formed microstructures at different stages. The visualized profile apparently indicated the filling behavior of glass flow during the imprinting process. Good agreement between FEM simulation and experimental results was achieved even though a slight difference between them was discovered because of calculation error from the experiment. As a result, comparison of the conditions, parameters, and results of FEM simulation with the experimental results can assist us profoundly in probing into many relative issues, such as the mechanism of microstructure formability on a glass substrate, the effect of process parameters on glass imprinting, and the pressure and velocity distribution of glass flow, which enable us to efficiently optimize the imprinting process.