The cross-linking with reducing sugars, known as glycation, is used to increase stiffness and strength of tissues and artificial collagen-based scaffolds. Nondestructive characterization methods that report on the structures within these materials could clarify the effects of glycation. For doing this nondestructive evaluation, we employed an in situ one-photon fluorescence as well as multiphoton microscopy method that combined two-photon fluorescence and second harmonic generation signals. We incubated collagen hydrogels with glyceraldehyde, ribose, and glucose and observed an increase in the in situ fluorescence and structural alterations within the materials during the course of glycation. The two-photon fluorescence emission maximum was observed at about 460 nm. The fluorescence emission in the one-photon excitation experiment (λ(ex) = 360 nm) was broad with peaks centered at 445 and 460 nm. The 460 nm emission component subsequently became dominant during the course of glycation with glyceraldehyde. For the ribose, in addition to the 460 nm peak, the 445 nm component persisted. The glucose glycated hydrogels exhibited broad fluorescence that did not increase significantly even after 6 weeks. As determined from measuring the fluorescence intensity at the 460 nm maximum, glycation with glyceraldehyde was faster compared to ribose and generated stronger fluorescence signals. Upon excitation of glycated samples with 330 nm light, different emission peaks were observed.